Platforms, compositions, and methods for therapeutic delivery

ABSTRACT

Descried herein are platforms for generating extracellular vesicles. Described herein are compositions of extracellular vesicles. Also described herein are methods of using the extracellular vesicles for therapeutics delivery.

CROSS-REFERENCE

This application is a Continuation of International Application No.PCT/US2020/038816, filed Jun. 19, 2020, which claims the benefit of U.S.Provisional Application Ser. No. 62/864,566 filed on Jun. 21, 2019 andU.S. Provisional Application Ser. No. 62/875,001 filed on Jul. 17, 2019,the entirety of which are hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 17, 2021, isnamed 58743-701_301_SL.txt, and is 40,984 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.This application incorporates “Riazifar M, Mohammadi M R, Pone E J, etal. Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune andNeurodegenerative Disorders. ACS Nano. 2019; 13(6):6670-6688.doi:10.1021/acsnano.9b01004”, the entirety of which are herebyincorporated by reference herein.

BACKGROUND

Effective means of delivering therapeutics to a target cell is one ofthe cornerstones of modern medicine. Extracellular vesicles have beenexplored and utilized as carriers for delivering therapeutics to atarget cell.

SUMMARY

Current methods of delivering therapeutics via extracellular vesicleshave drawbacks. The extracellular vesicles can be heterogenous in termsof sizes, numbers, membrane properties, or stability. The amount oftherapeutics that can delivered by the extracellular vesicles can beuneven or unpredictable, leading to ineffective treatment outcomes. Insome instances, the yield of the extracellular vesicles can beinadequate. Additionally, directing the extracellular vesicles to an invivo target cell is an ongoing challenge due to majority of theextracellular vesicles in circulation accumulate in liver, spleen, andkidney. Therefore, there remains a need for compositions andpharmaceutical compositions comprising extracellular vesicles fordelivering sufficient quantity of therapeutics to a target cell. Thereremains a need for platforms and methods for producing extracellularvesicles to deliver sufficient quantity of therapeutics to a targetcell. There also remains a need for methods of using extracellularvesicles to deliver therapeutics and methods of using extracellularvesicles to treat a disease or a condition.

An aspect of the present disclosure comprises a composition comprisingan extracellular vesicle, said extracellular vesicle comprising: animmune checkpoint moiety, wherein said immune checkpoint moietycomprises VISTA, PD-L1, CTLA-4, or any combination thereof; and atransmembrane moiety, wherein said transmembrane moiety comprises CD63,wherein said CD63 comprises 3 transmembrane domains; wherein said immunecheckpoint moiety is coupled to said transmembrane moiety. In someembodiments, said immune checkpoint moiety is coupled to anextracellular loop of said CD63 to generate a modified CD63. In someembodiments, said extracellular loop is a large extracellular loop orthe second extracellular loop of said modified CD63. In someembodiments, said modified CD63 comprises an amino acid sequence atleast 90% identical to any one of the amino acid sequences set forth inSEQ ID Nos: 6-17. In some embodiments, said modified CD63 comprises anamino acid sequence at least 95% identical to any one of the amino acidsequences set forth in SEQ ID Nos: 6-17. In some embodiments, saidmodified CD63 comprises any one of the amino acid sequences set forth inSEQ ID Nos: 6-17. A composition comprising an extracellular vesicle,said extracellular vesicle comprising: an immune checkpoint moiety,wherein said immune checkpoint moiety comprises PD-L1; and atransmembrane moiety, wherein said transmembrane moiety compriseslactadherin; wherein said immune checkpoint moiety is coupled to saidtransmembrane moiety. A composition comprising an extracellular vesicle,said extracellular vesicle comprising: an immune checkpoint moiety,wherein said immune checkpoint moiety comprises V-domain Ig suppressorof T cell activation (VISTA), PD-L1, CTLA-4, or any combination thereof;and a transmembrane moiety, wherein said transmembrane moiety comprisesglycosylphosphatidylinositol (GPI); wherein said immune checkpointmoiety is coupled to said transmembrane moiety. A composition comprisingan extracellular vesicle, said extracellular vesicle comprising at leastone of: an immune checkpoint moiety; and a transmembrane moiety. In someembodiments, the immune checkpoint moiety is encapsulated by theextracellular vesicle. In some embodiments, the immune checkpoint moietyis expressed on a surface of the extracellular vesicle. In someembodiments, the immune checkpoint moiety is secreted by theextracellular vesicle. In some embodiments, the immune checkpoint moietyis complexed with the transmembrane moiety. In some embodiments, theimmune checkpoint moiety is covalently connected with the transmembranemoiety. In some embodiments, the immune checkpoint moiety comprisesVISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276),B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3,CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112),FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1,Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L,OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3,CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha(CD172a), or a combination thereof. In some embodiments, the immunecheckpoint moiety comprises VISTA, PD-L1, CTLA-4, or a combinationthereof. In some embodiments, the immune checkpoint moiety comprisesPD-L1. In some embodiments, the transmembrane moiety is selected from agroup consisting of 14-3-3 protein zeta/delta, 4-3-3 protein epsilon, 78kDa glucose-regulated protein, acetylcholinesterase/AChE-S, AChE-E,actin, cytoplasmic 1 (ACTA), ADAM10, alkaline phosphatase,alpha-enolase, alpha-synuclein, aminopeptidase N, amyloid beta A4/APP,annexin 5A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, betaactin (ACTB), beta-amyloid 42, caveolin-1, CD10, CD11a, CD11b, CD11c,CD14, CD142, CD146, CD163, CD24, CD26/DPP4, CD29/ITGB1, CD3, CD37, CD41,CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73 CD81,CD82, CD9, CD90, claudin, claudin-1 cofilin-1, complement-bindingproteins CD55 and CD59, cytosolic heat shock protein 90 alpha, cytosolicheat shock protein 90 beta, EBV LMP1, EBV LMP2A, EF-1alpha-1, EF2, EFGREGFR VIII, emmprin/CD147, enolase 1 alpha (ENO1), EPCAM, ERBB2,tetraspanins (CD9, CD63 and CD81), fatty acid synthase, fetuin-A,flotillin-1, flotillin-2, fructose-bisphosphate aldolase A,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin A, GPC1,GPI-anchored 5′nucleotidase, GTPase, heat shock protein 8 (HSPA8), heatshock proteins (HSP70 and HSP90), heparan sulfate proteoglycans,heparinase, heterotrimeric G proteins, HIV Gag, HIV Nef, HLA-DRA, HLA-G,HSV gB, HTLV-1 Tax, huntingtin, ICAM1, integrins, lactadherin, LAMP1/2,leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain,lysosome-associated membrane glycoprotein 1, lysosome-associatedmembrane glycoprotein 2, MEW class I, MHC class II, MUC1, multidrugresistance-associated protein, muscle pyruvate kinase (PKM2),N-cadherin, NKCC2, PDCD6IP/Alix, PECAM1, phosphoglycerate kinase,placental prion proteins, prostate-specific antigen (PSA), pyruvatekinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap 1B, resistin,sonic hedgehog (SHH), surviving, syndecan-1, syndecan-4, syntenin-1,transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associatedglycoprotein tetraspanin-8, tyrosine 3 monooxygenase/tryptophan5-monooxygenase activation protein, TYRP-2, vacuolar-sorting protein 35,or zeta polypeptide (YWHAZ). In some embodiments, the transmembranemoiety comprises lactadherin. In some embodiments, the transmembranemoiety comprises LAMP2 or a variation thereof or a fragment thereof,said LAMP2 is at least 70% identical to a peptide sequence of SEQ ID NO:4 In some embodiments, the transmembrane moiety comprises CD63 or avariation thereof or a fragment thereof, said CD63 is at least 70%identical to a peptide sequence of SEQ ID NO: 5. In some embodiments,the CD63 is a modified CD63. In some embodiments, the modified CD63 is atruncated CD63. In some embodiments, the modified CD63 is modified tocomprise at least one additional CD63 transmembrane domain. In someembodiments, the modified CD63 comprises 1 transmembrane domain. In someembodiments, the modified CD63 comprises 2 transmembrane domains. Insome embodiments, the modified CD63 comprises 3 transmembrane domains.In some embodiments, the modified CD63 comprises 4 transmembranedomains. In some embodiments, the modified CD63 comprises 5transmembrane domains. In some embodiments, the immune checkpoint moietyis complexed with the modified CD63 at an extracellular loop of themodified CD63. In some embodiments, the immune checkpoint moiety iscomplexed with the modified CD63 at a large extracellular loop of themodified CD63. In some embodiments, the composition further comprises atargeting moiety. In some embodiments, the targeting moiety comprises apeptide that targets cytokine. In some embodiments, the targeting moietycomprises a peptide that targets a cancer cell marker. In someembodiments, the composition further comprises a fusogenic moiety. Insome embodiments, the fusogenic moiety comprises a viral fusogenicmoiety. In some embodiments, the fusogenic moiety comprises a mammalianfusogenic moiety. In some embodiments, the composition further comprisesan immune evasion moiety. In some embodiments, the immune evasion moietycomprises CD47. The composition of any one of the previous claims doesnot comprise enucleated cell. In some embodiments, the extracellularvesicle comprises exosome, microvesicle, retrovirus-like particle,apoptotic body, apoptosome, oncosome, exopher, enveloped virus, exomere,or other very large extracellular vesicle. In some embodiments, theextracellular vesicle comprises exosome. In some embodiments, theextracellular vesicle comprises a plurality of immune checkpointmoieties. In some embodiments, the extracellular vesicle comprises atleast 10,000 units of the immune checkpoint moiety per eachextracellular vesicle, said extracellular vesicle comprises a diameterof 100 nm. In some embodiments, the extracellular vesicle comprises atleast 9,000 units of the immune checkpoint moiety per each extracellularvesicle, said extracellular vesicle comprises a diameter of 100 nm. Insome embodiments, the extracellular vesicle comprises at least 8,000units of the immune checkpoint moiety per each extracellular vesicle,said extracellular vesicle comprises a diameter of 100 nm. In someembodiments, the extracellular vesicle comprises at least 7,000 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 100 nm. In someembodiments, the extracellular vesicle comprises at least 6,000 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 100 nm. In someembodiments, the extracellular vesicle comprises at least 5,000 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 100 nm. In someembodiments, the extracellular vesicle comprises at least 3,000 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 50 nm. In someembodiments, the extracellular vesicle comprises at least 2,500 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 50 nm. In someembodiments, the extracellular vesicle comprises at least 2,000 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 50 nm. In someembodiments, the extracellular vesicle comprises at least 1,500 units ofthe immune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 50 nm. In someembodiments, the composition comprises a plurality of exosome. In someembodiments, the composition comprises a therapeutically effectiveamount of exosome. In some embodiments, the composition furthercomprises at least 10{circumflex over ( )}6 exosomes. In someembodiments, the composition further comprises at least 10{circumflexover ( )}7 exosomes. In some embodiments, the composition furthercomprises at least 10{circumflex over ( )}8 exosomes. In someembodiments, the composition further comprises at least 1 μg ofexosomes. In some embodiments, the composition further comprises atleast 10 μg of exosomes. In some embodiments, the composition furthercomprises at least 20 μg of exosomes. In some embodiments, thecomposition further comprises at least 50 μg of exosomes. In someembodiments, the composition further comprises at least 100 μg ofexosomes. In some embodiments, the composition further comprises atleast 150 μg of exosomes. In some embodiments, the composition furthercomprises at least 200 μg of exosomes. In some embodiments, thecomposition further comprises at least 250 μg of exosomes. In someembodiments, the composition further comprises at least 500 μg ofexosomes. In some embodiments, the composition further comprises atleast 750 μg of exosomes. In some embodiments, the composition furthercomprises at least 1 mg of exosomes. In some embodiments, thecomposition further comprises at least 2 mg of exosomes. In someembodiments, the composition further comprises at least 3 mg ofexosomes. In some embodiments, the composition further comprises atleast 4 mg of exosomes. In some embodiments, the composition furthercomprises at least 5 mg of exosomes. In some embodiments, thecomposition further comprises at least 6 mg of exosomes. In someembodiments, the composition further comprises at least 7 mg ofexosomes. In some embodiments, the composition further comprises atleast 100 mg of exosomes. In some embodiments, the composition furthercomprises at least 200 mg of exosomes. In some embodiments, thecomposition further comprises at least 300 mg of exosomes. In someembodiments, the composition further comprises at least 400 mg ofexosomes. In some embodiments, the composition further comprises atleast 500 mg of exosomes. In some embodiments, the composition furthercomprises at least 600 mg of exosomes. In some embodiments, thecomposition further comprises at least 700 mg of exosomes. In someembodiments, the composition is derived from a cell. In someembodiments, the composition is cryopreserved. In some embodiments, thecomposition is lyophilized. In some embodiments, the composition isstable at 37° C. for 24 hours. In some embodiments, the composition isstable at 37° C. for 48 hours. In some embodiments, the composition isstable at 37° C. for 72 hours.

Another aspect of the present disclosure comprises a cell configured togenerate the extracellular vesicle or exosome of any one of theembodiments described herein. In some embodiments, said cell is a stemcell. In some embodiments, wherein the cell is a human cell. In someembodiments, the cell is a non-human cell. In some embodiments, the cellis a mesenchymal stem cell. In some embodiments, the cell is agenetically modified cell. In some embodiments, the cell is geneticallymodified to produce the extracellular vesicles or exosomes described inany one of the embodiments described herein.

Another aspect of the present disclosure comprises a method of purifyingextracellular vesicle configured to express one or more immunecheckpoint moiety, said method comprising: obtaining a heterogenouspopulation of extracellular vesicle; subjecting the heterogenouspopulation of extracellular vesicle to a detection assay solution, saiddetection assay solution comprising a detecting moiety for complexingwith the immune checkpoint moiety; and detecting a signal generated fromcomplex formed between the immune checkpoint moiety and the detectingmoiety, wherein intensity of the signal is proportional to units ofimmune checkpoint moiety expressed; and isolating a subpopulation of theextracellular vesicle based on the intensity of the signal. In someembodiments, the detecting moiety comprises an antibody. In someembodiments, the detecting moiety comprises anti-VISTA antibody,anti-PD-L1 antibody, anti-CTLA-4 or a combination thereof. In someembodiments, the detecting moiety comprises a ligand of the immunecheckpoint moiety. In some embodiments, the detection assay solutionfurther comprises a peptide configured to bind to said immune checkpointmoiety. In some embodiments, the peptide configured to bind to saidimmune checkpoint moiety comprises PD-1, CD80, CD86, or a combinationthereof.

Another aspect of the present disclosure comprises a pharmaceuticalcomposition comprising the composition of any one of the embodimentsdescribed herein. In some embodiments, the pharmaceutical compositionfurther comprises a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition further comprises at leastone additional active agent. In some embodiments, the pharmaceuticalcomposition is formulated for administering intrathecally,intraocularly, intravitreally, retinally, intravenously,intramuscularly, intraventricularly, intracerebrally, intracerebellarly,intracerebroventricularly, intraperenchymally, subcutaneously, or acombination thereof.

Another aspect of the present disclosure comprises a method of treatingan autoimmune disease, the method comprising administering thepharmaceutical composition of any one of the embodiments describedherein. In some embodiments, said autoimmune disease is Rheumatoidarthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetesmellitus, Multiple sclerosis, Inflammatory bowel disease, Celiacdisease, Crohn's disease, Graves' disease, Juvenile arthritis, Lymedisease chronic, Optic neuritis, Psoriatic arthritis, Scleritis,Scleroderma, Ulcerative colitis (UC), Uveitis, Inflammatory eyeconditions, Vitiligo, COPD, complication from Organ transplantation, orgraft-versus-host disease. In some embodiments, said autoimmune diseaseis Rheumatoid arthritis.

Another aspect of the present disclosure comprises a method ofsuppressing CD8+CD25+ cells in a patient in need thereof, the methodcomprising administering the composition of any one of the embodimentsdescribed herein.

Another aspect of the present disclosure comprises a kit comprising thepharmaceutical composition of any one of the embodiments describedherein.

Another aspect of the present disclosure comprises a platform comprisingcomponents for generating the composition of any one of the embodimentsdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application contains at least one drawing executed in color.Copies of this patent or patent application with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 illustrates proteomics analysis on the human MSC-derived exosomesdetecting. Programmed death ligand-1 (PD-L1).

FIG. 2 illustrates an experimental design for blocking PD-L1 todetermine how blocking PD-L1 affected CD8+ T cells.

FIG. 3 illustrates that the presence of blocking PD-L1 antibodyabolished the effected of PD-L1 on CD25+CD8+ cell.

DETAILED DESCRIPTION

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

Use of absolute or sequential terms, for example, “will,” “will not,”“shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,”“subsequently,” “before,” “after,” “lastly,” and “finally,” are notmeant to limit scope of the present embodiments disclosed herein but asexemplary.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As used herein, the phrases “at least one”, “one or more”, and “and/or”are open-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “oneor more of A, B, or C”, and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

Any systems, methods, software, and platforms described herein aremodular and not limited to sequential steps. Accordingly, terms such as“first” and “second” do not necessarily imply priority, order ofimportance, or order of acts.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

The terms “increased” or “increase” are used herein to generally mean anincrease by a statically significant amount; in some embodiments, theterms “increased,” or “increase,” mean an increase of at least 10% ascompared to a reference level, for example an increase of at least about10%, at least about 20%, or at least about 30%, or at least about 40%,or at least about 50%, or at least about 60%, or at least about 70%, orat least about 80%, or at least about 90%, or up to and including a 100%increase, or any increase between 10-100% as compared to a referencelevel, standard, or control. Other examples of “increase” include anincrease of at least 2-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, ormore as compared to a reference level.

The terms “decreased” or “decrease” are used herein generally to mean adecrease by a statistically significant amount. In some embodiments,“decreased” or “decrease” means a reduction by at least 10% as comparedto a reference level, for example a decrease by at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90%, or up to and including a 100% decrease (e.g., absentlevel or non-detectable level as compared to a reference level), or anydecrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom, by these terms is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without a given disease.

The terms “individual,” “patient,” or “subject” are usedinterchangeably. None of the terms require or are limited to situationcharacterized by the supervision (e.g., constant or intermittent) of ahealth care worker (e.g., a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

As used herein, a “cell” generally refers to a biological cell. A cellcan be the basic structural, functional and/or biological unit of aliving organism. A cell can originate from any organism having one ormore cells. Some non-limiting examples include: a prokaryotic cell,eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g., cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin,hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers,gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algalcell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii,Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C.Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., ayeast cell, a cell from a mushroom), an animal cell, a cell from aninvertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, asheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.),and etcetera. Sometimes a cell is not originating from a naturalorganism (e.g., a cell can be a synthetically made, sometimes termed anartificial cell).

The term “nucleotide,” as used herein, generally refers to abase-sugar-phosphate combination. A nucleotide can comprise a syntheticnucleotide. A nucleotide can comprise a synthetic nucleotide analog.Nucleotides can be monomeric units of a nucleic acid sequence (e.g.,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The termnucleotide can include ribonucleoside triphosphates adenosinetriphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate(CTP), guanosine triphosphate (GTP) and deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives can include, for example, [αS]dATP,7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confernuclease resistance on the nucleic acid molecule containing them. Theterm nucleotide as used herein can refer to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrative examples ofdideoxyribonucleoside triphosphates can include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled ordetectably labeled by well-known techniques. Labeling can also becarried out with quantum dots. Detectable labels can include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels. Fluorescent labels ofnucleotides can include but are not limited fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides can include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif.; FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.Nucleotides can also be labeled or marked by chemical modification. Achemically-modified single nucleotide can be biotin-dNTP. Somenon-limiting examples of biotinylated dNTPs can include, biotin-dATP(e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP,biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP,biotin-20-dUTP).

The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” areused interchangeably to refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof, either in single-, double-, or multi-stranded form. Apolynucleotide can be exogenous or endogenous to a cell. Apolynucleotide can exist in a cell-free environment. A polynucleotidecan be a gene or fragment thereof. A polynucleotide can be DNA. Apolynucleotide can be RNA. A polynucleotide can have any threedimensional structure, and can perform any function, known or unknown. Apolynucleotide can comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). If present, modifications to the nucleotidestructure can be imparted before or after assembly of the polymer. Somenon-limiting examples of analogs include: 5-bromouracil, peptide nucleicacid, xeno nucleic acid, morpholinos, locked nucleic acids, glycolnucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin,7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to thesugar), thiol containing nucleotides, biotin linked nucleotides,fluorescent base analogs, CpG islands, methyl-7-guanosine, methylatednucleotides, inosine, thiouridine, pseudourdine, dihydrouridine,queuosine, and wyosine. Non-limiting examples of polynucleotides includecoding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,cell-free polynucleotides including cell-free DNA (cfDNA) and cell-freeRNA (cfRNA), nucleic acid probes, and primers. The sequence ofnucleotides can be interrupted by non-nucleotide components.

The terms “transfection” or “transfected” generally refers tointroduction of a nucleic acid into a cell by non-viral or viral-basedmethods. The nucleic acid molecules can be gene sequences encodingcomplete proteins or functional portions thereof. See, e.g., Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

The term “expression” or “expressing” refers to one or more processes bywhich a polynucleotide is transcribed from a DNA template (such as intoan mRNA or other RNA transcript) and/or the process by which atranscribed mRNA is subsequently translated into peptides, polypeptides,or proteins. Transcripts and encoded polypeptides can be collectivelyreferred to as “gene product.” If the polynucleotide is derived fromgenomic DNA, expression can include splicing of the mRNA in a eukaryoticcell. “Up-regulated,” with reference to expression, generally refers toan increased expression level of a polynucleotide (e.g., RNA such asmRNA) and/or polypeptide sequence relative to its expression level in awild-type state while “down-regulated” generally refers to a decreasedexpression level of a polynucleotide (e.g., RNA such as mRNA) and/orpolypeptide sequence relative to its expression in a wild-type state.

The term “gene,” as used herein, refers to a segment of nucleic acidthat encodes an individual protein or RNA (also referred to as a “codingsequence” or “coding region”), optionally together with associatedregulatory region such as promoter, operator, terminator and the like,which can be located upstream or downstream of the coding sequence. Theterm “gene” is to be interpreted broadly, and can encompass mRNA, cDNA,cRNA and genomic DNA forms of a gene. In some uses, the term “gene”encompasses the transcribed sequences, including 5′ and 3′ untranslatedregions (5′-UTR and 3′-UTR), exons and introns. In some genes, thetranscribed region will contain “open reading frames” that encodepolypeptides. In some uses of the term, a “gene” comprises only thecoding sequences (e.g., an “open reading frame” or “coding region”)necessary for encoding a polypeptide. In some aspects, genes do notencode a polypeptide, for example, ribosomal RNA genes (rRNA) andtransfer RNA (tRNA) genes. In some aspects, the term “gene” includes notonly the transcribed sequences, but in addition, also includesnon-transcribed regions including upstream and downstream regulatoryregions, enhancers and promoters. The term “gene” can encompass mRNA,cDNA and genomic forms of a gene.

As used herein, the terms “polypeptide,” “peptide” and “protein” can beused interchangeably herein in reference to a polymer of amino acidresidues. A protein can refer to a full-length polypeptide as translatedfrom a coding open reading frame, or as processed to its mature form,while a polypeptide or peptide can refer to a degradation fragment or aprocessing fragment of a protein that nonetheless uniquely oridentifiably maps to a particular protein. A polypeptide can be a singlelinear polymer chain of amino acids bonded together by peptide bondsbetween the carboxyl and amino groups of adjacent amino acid residues.Polypeptides can be modified, for example, by the addition ofcarbohydrate, phosphorylation, etc. Proteins can comprise one or morepolypeptides.

As used herein, the terms “fragment,” or equivalent terms can refer to aportion of a protein that has less than the full length of the proteinand optionally maintains the function of the protein. Further, when theportion of the protein is blasted against the protein, the portion ofthe protein sequence can align, for example, at least with 80% identityto a part of the protein sequence.

The terms “complement,” “complements,” “complementary,” and“complementarity,” as used herein, generally refer to a sequence that isfully complementary to and hybridizable to the given sequence. In somecases, a sequence hybridized with a given nucleic acid is referred to asthe “complement” or “reverse-complement” of the given molecule if itssequence of bases over a given region is capable of complementarilybinding those of its binding partner, such that, for example, A-T, A-U,G-C, and G-U base pairs are formed. In general, a first sequence that ishybridizable to a second sequence is specifically or selectivelyhybridizable to the second sequence, such that hybridization to thesecond sequence or set of second sequences is preferred (e.g.,thermodynamically more stable under a given set of conditions, such asstringent conditions commonly used in the art) to hybridization withnon-target sequences during a hybridization reaction. Typically,hybridizable sequences share a degree of sequence complementarity overall or a portion of their respective lengths, such as between 25%-100%complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, and 100% sequence complementarity. Sequence identity, such asfor the purpose of assessing percent complementarity, can be measured byany suitable alignment algorithm, including but not limited to theNeedleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligneravailable at www.ebi.ac.uk/Tools/psa/emboss needle/nucleotide.html,optionally with default settings), the BLAST algorithm (see e.g. theBLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi,optionally with default settings), or the Smith-Waterman algorithm (seee.g. the EMBOSS Water aligner available atwww.ebi.ac.uk/Tools/psa/emboss water/nucleotide.html, optionally withdefault settings). Optimal alignment can be assessed using any suitableparameters of a chosen algorithm, including default parameters.

The term “percent (%) identity,” as used herein, generally refers to thepercentage of amino acid (or nucleic acid) residues of a candidatesequence that are identical to the amino acid (or nucleic acid) residuesof a reference sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent identity (i.e., gapscan be introduced in one or both of the candidate and referencesequences for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). Alignment, for purposes ofdetermining percent identity, can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.Percent identity of two sequences can be calculated by aligning a testsequence with a comparison sequence using BLAST, determining the numberof amino acids or nucleotides in the aligned test sequence that areidentical to amino acids or nucleotides in the same position of thecomparison sequence, and dividing the number of identical amino acids ornucleotides by the number of amino acids or nucleotides in thecomparison sequence.

The term “mismatch” generally refers to lack of complementarity betweentwo nucleotides when aligned. Complementary bases in DNA are A-T andG-C. Complementary bases in RNA are A-U and G-C. Thus a mismatch occurswhen two oligonucleotide sequences are aligned and at one or morenucleotide positions that an A is not paired with T or a G is not pairedwith C in DNA or an A is not paired with U or a G is not paired with Cin RNA.

As used herein, the term “in vivo” can be used to describe an event thattakes place in a subject's body.

As used herein, the term “ex vivo” can be used to describe an event thattakes place outside of a subject's body. An “ex vivo” assay cannot beperformed on a subject. Rather, it can be performed upon a sampleseparate from a subject. Ex vivo can be used to describe an eventoccurring in an intact cell outside a subject's body.

As used herein, the term “in vitro” can be used to describe an eventthat takes places contained in a container for holding laboratoryreagent such that it is separated from the living biological sourceorganism from which the material is obtained. In vitro assays canencompass cell-based assays in which cells alive or dead are employed.In vitro assays can also encompass a cell-free assay in which no intactcells are employed.

“Treating” or “treatment” can refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) a targeted pathologic condition or disorder. Thosein need of treatment include those already with the disorder, as well asthose prone to have the disorder, or those in whom the disorder is to beprevented. A therapeutic benefit can refer to eradication oramelioration of symptoms or of an underlying disorder being treated.Also, a therapeutic benefit can be achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thesubject, notwithstanding that the subject can still be afflicted withthe underlying disorder. A prophylactic effect can include delaying,preventing, or eliminating the appearance of a disease or condition,delaying or eliminating the onset of symptoms of a disease or condition,slowing, halting, or reversing the progression of a disease orcondition, or any combination thereof. For prophylactic benefit, asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease canundergo treatment, even though a diagnosis of this disease cannot havebeen made.

The term “effective amount” and “therapeutically effective amount,” asused interchangeably herein, generally refer to the quantity of acomposition, for example a composition comprising immune cells such aslymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system ofthe present disclosure, that is sufficient to result in a desiredactivity upon administration to a subject in need thereof. Within thecontext of the present disclosure, the term “therapeutically effective”refers to that quantity of a composition that is sufficient to delay themanifestation, arrest the progression, relieve or alleviate at least onesymptom of a disorder treated by the methods of the present disclosure.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial. A component can be “pharmaceutically acceptable” in the senseof being compatible with the other ingredients of a pharmaceuticalformulation. It can also be suitable for use in contact with the tissueor organ of humans and animals without excessive toxicity, irritation,allergic response, immunogenicity, or other problems or complications,commensurate with a reasonable benefit/risk ratio. See, Remington: TheScience and Practice of Pharmacy, 21st Edition; Lippincott Williams &Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients,5th Edition”; Rowe et al., Eds., The Pharmaceutical Press and theAmerican Pharmaceutical Association: 2005; and Handbook ofPharmaceutical Additives, 3rd Edition; Ash and Ash Eds., GowerPublishing Company: 2007; Pharmaceutical Preformulation and Formulation,Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition can facilitate administrationof the compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.

The instant disclosure is directed to mesenchymal stem cell exosomesengineered with PD-L1: a novel cell free therapeutics to inducetolerance in autoimmune diseases and transplantation. Extracellularvesicles, such as exosomes and microvesicles (also known as sheddingvesicles), carry bioactive molecules that influence the extracellularenvironment and the immune system. It has been shown that exosomal PD-L1has the same membrane topology as cell surface PD-L1, with itsextracellular domain exposed on the surface of the exosomes. ExosomalPD-L1 binds PD-1 in a concentration-dependent manner, and thisinteraction can be disrupted by PD-L1-blocking antibodies. High levelsof exosomal PD-L1 may reflect the ‘exhaustion’ of T cells to a stage atwhich they can no longer be reinvigorated by anti-PD-1 treatment.Moreover, increase in exosomal PD-L1 in response to IFN-γ could enabletumour cells to adaptively inactivate CD8 T cells.

Mesenchymal stem is first isolated from healthy individuals. Onceexpanded, these cells can be stored for further use. These cells canthen be engineered with PD-L1. To this end, PDL1 gene can be fused withLactadherin gene. This allows the PD-L1 molecule to be inserted outsidethe exosomes. Upon confirmation of successful engineering, these cellsare grown on bioreactor and conditioned media can be harvested toisolate exosomes. For exosome isolation, tangential flow filtration willbe used to harvest and purify exosomes. These exosomes have the abilityto suppress immune system in particular CD8+ and CD25+ effector cells.These effector cells are the key in pathogenesis of all autoimmunediseases and rejection processes in transplantation. Once these exosomesare generated under cGMP facilities and pass all potency and otherrequirements, they can be used to treat patients. In some embodiments,the mesenchymal stem cells exosomes can be engineered with PD-L1 toenhance their suppressive activity. In some embodiments, the PD-L1engineered mesenchymal stem cell exosomes can be a treatment for allautoimmune diseases and transplantation including Type I diabetes,Multiple Sclerosis, COPD, Lupus, inflammatory bowel disease, Rheumatoidarthritis, or Psoriasis. In some embodiments, the function of the PD-L1engineered mesenchymal stem cell exosome can be increased if engineeredregulatory T cells are cultured under hypoxia conditions. In someembodiments, the PD-L1 engineered mesenchymal stem cell exosome can havethe ability to suppress activated immune system in particular, CD8+CD25+cells in patients. In some embodiments, using the platform describedherein, any other cells or exosomes can be engineered to comprise PD-L1,similar to mesenchymal stem cell and mesenchymal stem cell derivedexosomes. In some embodiments, the PD-L1 engineered mesenchymal stemcell exosomes can be prepared under cGMP facility. In some embodiments,the PD-L1 engineered mesenchymal stem cell exosomes can be considered asclinical-grade exosomes to treat all autoimmune disease and patients whoreceived any organ as transplantation procedures.

The instant disclosure is directed to mesenchymal stem cell exosomesengineered with VISTA/PD-L1/CTLA-4: A novel cell free therapeutics toinduce tolerance in autoimmune diseases and transplantation.Extracellular vesicles, such as exosomes and microvesicles (also knownas shedding vesicles), carry bioactive molecules that influence theextracellular environment and the immune system. It has been shown thatexosomal PD-L1 has the same membrane topology as cell surface PD-L1,with its extracellular domain exposed on the surface of the exosomes.Exosomal PD-L1 binds PD-1 in a concentration-dependent manner, and thisinteraction can be disrupted by PD-L1-blocking antibodies. High levelsof exosomal PD-L1 may reflect the “exhaustion” of T cells to a stage atwhich they can no longer be reinvigorated by anti-PD-1 treatment.Moreover, increase in exosomal PD-L1 in response to IFN-γ can enabletumour cells to adaptively inactivate CD8 T cells. V-domain Igsuppressor of T cell activation (VISTA) is a potent negative regulatorof T-cell function that is expressed on hematopoietic cells. VISTAlevels are heightened within the tumor microenvironment, in which itsblockade can enhance antitumor immune responses in mice. VISTA isexpressed predominantly within the hematopoietic compartment withhighest expression within the myeloid lineage. VISTA-Ig suppressedproliferation of T cells and blunted the production of T-cell cytokinesand activation markers. VISTA is an inhibitory immune-checkpointmolecule that suppresses CD4+ and CD8+ T cell activation when expressedon antigen-presenting cells. VISTA critically regulates the inflammatoryresponses mediated by DCs and IL-17-producing TCRγδ+ and CD4+Th17 Tcells following TLR7 stimulation. CTLA4 or CTLA-4 (cytotoxicT-lymphocyte-associated protein 4), also known as CD152 (cluster ofdifferentiation 152), is a protein receptor that functions as an immunecheckpoint and downregulates immune responses. CTLA4 is constitutivelyexpressed in regulatory T cells but only upregulated in conventional Tcells after activation—a phenomenon which is particularly notable incancers. It acts as an “off” switch when bound to CD80 or CD86 on thesurface of antigen-presenting cells.

In some embodiments, mesenchymal stem cell can be first isolated fromhealthy individuals. Once expanded, these cells can be stored forfurther use. These cells can then be engineered with VISTA/PD-L1/CTLA-4alone or in combination. The following immune check points can be usedinstead or in combination of VISTA, PD-L1 and CTLA-4: PD-L2, B7-1(CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270,TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR(CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47,Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2(CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4(CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3(CD223), PECAM-1, CD44, or SIRP alpha (CD172a).

To this end, one or combination of the immune check point genesdescribed herein can be fused with Lactadherin gene or GPI proteins orother protein or linkers. This allows the check points protein to beinserted outside the exosomes. Upon confirmation of successfulengineering, these cells are grown on bioreactor and conditioned mediacan be harvested to isolate exosomes. For exosome isolation, tangentialflow filtration can be used to harvest and purify exosomes. Theseexosomes have the ability to suppress immune system in particularCD4+/CD25+; CD8+CD25+ effector cells or dendritic cells and B cells.This suppression depends on combination of check point molecules on thesurface of exosomes. These effector cells are the key in pathogenesis ofall autoimmune diseases and rejection processes in transplantation. Oncethese exosomes are generated under cGMP facilities and pass all potencyand other requirements, they can be used to treat patients. In someembodiments, mesenchymal stem cells exosomes can be engineered withcheck point molecules to enhance their suppressive activity. This checkpoint can be combination of VISTA/PD-L1/CTLA-4 or alone. In someembodiments, the check point can be any combination of the immune checkpoint genes described herein. In some embodiments, the check pointdecorated mesenchymal stem cell exosomes can be a treatment for allautoimmune diseases and transplantation including but not limited torheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1diabetes mellitus, multiple sclerosis, inflammatory bowel disease,celiac disease, Crohn's disease, Graves' disease, juvenile arthritis,Lyme disease, optic neuritis, psoriatic arthritis, scleritis,scleroderma, ulcerative colitis (UC), uveitis, inflammatory eyeconditions, vitiligo, COPD, or organ transplantation. In someembodiments, any other cells or exosomes can be engineered with theimmune check point (similar to mesenchymal stem cell and their exosomesof human fibroblast) by utilizing the platforms described herein. Insome embodiments, decorated exosomes with check point molecules can bemanufactured as injectable, eye drop, nebulizer or spray, cream andtopical ointment or any other form 30 accepted as a medication. In someembodiments, there exosomes can be prepared under cGMP facility and canbe considered as clinical-grade exosomes to treat all autoimmune diseaseand patients who received any organ as transplantation procedures.

I. Compositions

Described herein, in some embodiments, is a composition comprisingextracellular vesicle generated from the platforms and methods describedherein. In some embodiments, the extracellular vesicle is amembrane-bound particle secreted by a cell. In some embodiments, theextracellular vesicle is a membrane-bound particle generated in vitro.In some embodiments, the extracellular vesicle is a membrane-boundparticle generated ex vivo. In some embodiments, the extracellularvesicle is a membrane-bound particle generated without a cell. In someembodiments, the extracellular vesicle is exosome, microvesicle,retrovirus-like particle, apoptotic body, apoptosome, oncosome, exopher,enveloped viruses, exomere, or other very large extracellular vesicle.In some embodiments, the extracellular vesicle is exosome.

In some instances, the extracellular vesicle comprises a diameter about1 nm to about 10,000 nm. In some instances, the extracellular vesiclecomprises a diameter about 1 nm to about 5 nm, about 1 nm to about 10nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm toabout 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm,about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nmto about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm toabout 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm,about 5 nm to about 1,000 nm, about 5 nm to about 2,000 nm, about 5 nmto about 5,000 nm, about 5 nm to about 10,000 nm, about 10 nm to about20 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10nm to about 200 nm, about 10 nm to about 500 nm, about 10 nm to about1,000 nm, about 10 nm to about 2,000 nm, about 10 nm to about 5,000 nm,about 10 nm to about 10,000 nm, about 20 nm to about 50 nm, about 20 nmto about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 500nm, about 20 nm to about 1,000 nm, about 20 nm to about 2,000 nm, about20 nm to about 5,000 nm, about 20 nm to about 10,000 nm, about 50 nm toabout 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm,about 50 nm to about 1,000 nm, about 50 nm to about 2,000 nm, about 50nm to about 5,000 nm, about 50 nm to about 10,000 nm, about 100 nm toabout 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000nm, about 100 nm to about 2,000 nm, about 100 nm to about 5,000 nm,about 100 nm to about 10,000 nm, about 200 nm to about 500 nm, about 200nm to about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm toabout 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm,about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm,about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, orabout 5,000 nm to about 10,000 nm. In some instances, the extracellularvesicle comprises a diameter about 1 nm, about 5 nm, about 10 nm, about20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In someinstances, the extracellular vesicle comprises a diameter at least about1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm,about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about5,000 nm. In some instances, the extracellular vesicle comprises adiameter at most about 5 nm, about 10 nm, about 20 nm, about 50 nm,about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000nm, about 5,000 nm, or about 10,000 nm.

In some embodiments, the extracellular vesicle comprises a diameter thatis at least about 1 nm to about 10,000 nm. In some embodiments, theextracellular vesicle comprises a diameter that is at least about 1 nmto about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm,about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm toabout 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm,about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nmto about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm toabout 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm,about 5 nm to about 2,000 nm, about 5 nm to about 5,000 nm, about 5 nmto about 10,000 nm, about 10 nm to about 20 nm, about 10 nm to about 50nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10nm to about 500 nm, about 10 nm to about 1,000 nm, about 10 nm to about2,000 nm, about 10 nm to about 5,000 nm, about 10 nm to about 10,000 nm,about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm toabout 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000nm, about 20 nm to about 2,000 nm, about 20 nm to about 5,000 nm, about20 nm to about 10,000 nm, about 50 nm to about 100 nm, about 50 nm toabout 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000nm, about 50 nm to about 2,000 nm, about 50 nm to about 5,000 nm, about50 nm to about 10,000 nm, about 100 nm to about 200 nm, about 100 nm toabout 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about2,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 10,000nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nmto about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm toabout 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about10,000 nm. In some embodiments, the extracellular vesicle comprises adiameter that is at least about 1 nm, about 5 nm, about 10 nm, about 20nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In someembodiments, the extracellular vesicle comprises a diameter that is atleast at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about2,000 nm, or about 5,000 nm. In some embodiments, the extracellularvesicle comprises a diameter that is at least at most about 5 nm, about10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

In some embodiments, the composition comprises a heterogenous populationof a plurality of extracellular vesicles. In some embodiments, theheterogeneous population of the extracellular vesicles comprises adiameter between about 1 nm to about 10,000 nm. In some embodiments, theheterogeneous population of the extracellular vesicles comprises adiameter between about 1 nm to about 5 nm, about 1 nm to about 10 nm,about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm toabout 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm,about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nmto about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm toabout 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm,about 5 nm to about 1,000 nm, about 5 nm to about 2,000 nm, about 5 nmto about 5,000 nm, about 5 nm to about 10,000 nm, about 10 nm to about20 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10nm to about 200 nm, about 10 nm to about 500 nm, about 10 nm to about1,000 nm, about 10 nm to about 2,000 nm, about 10 nm to about 5,000 nm,about 10 nm to about 10,000 nm, about 20 nm to about 50 nm, about 20 nmto about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 500nm, about 20 nm to about 1,000 nm, about 20 nm to about 2,000 nm, about20 nm to about 5,000 nm, about 20 nm to about 10,000 nm, about 50 nm toabout 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm,about 50 nm to about 1,000 nm, about 50 nm to about 2,000 nm, about 50nm to about 5,000 nm, about 50 nm to about 10,000 nm, about 100 nm toabout 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000nm, about 100 nm to about 2,000 nm, about 100 nm to about 5,000 nm,about 100 nm to about 10,000 nm, about 200 nm to about 500 nm, about 200nm to about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm toabout 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm,about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm,about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, orabout 5,000 nm to about 10,000 nm. In some embodiments, theheterogeneous population of the extracellular vesicles comprises adiameter between about 1 nm, about 5 nm, about 10 nm, about 20 nm, about50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about2,000 nm, about 5,000 nm, or about 10,000 nm. In some embodiments, theheterogeneous population of the extracellular vesicles comprises adiameter between at least about 1 nm, about 5 nm, about 10 nm, about 20nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000nm, about 2,000 nm, or about 5,000 nm. In some embodiments, theheterogeneous population of the extracellular vesicles comprises adiameter between at most about 5 nm, about 10 nm, about 20 nm, about 50nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about2,000 nm, about 5,000 nm, or about 10,000 nm.

In some embodiments, the heterogeneous population of the extracellularvesicles comprises a diameter that is at least about 1 nm to about10,000 nm. In some embodiments, the heterogeneous population of theextracellular vesicles comprises a diameter that is at least about 1 nmto about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm,about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm toabout 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm,about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nmto about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm toabout 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm,about 5 nm to about 2,000 nm, about 5 nm to about 5,000 nm, about 5 nmto about 10,000 nm, about 10 nm to about 20 nm, about 10 nm to about 50nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10nm to about 500 nm, about 10 nm to about 1,000 nm, about 10 nm to about2,000 nm, about 10 nm to about 5,000 nm, about 10 nm to about 10,000 nm,about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm toabout 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000nm, about 20 nm to about 2,000 nm, about 20 nm to about 5,000 nm, about20 nm to about 10,000 nm, about 50 nm to about 100 nm, about 50 nm toabout 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000nm, about 50 nm to about 2,000 nm, about 50 nm to about 5,000 nm, about50 nm to about 10,000 nm, about 100 nm to about 200 nm, about 100 nm toabout 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about2,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 10,000nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nmto about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm toabout 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about10,000 nm. In some embodiments, the heterogeneous population of theextracellular vesicles comprises a diameter that is at least about 1 nm,about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, orabout 10,000 nm. In some embodiments, the heterogeneous population ofthe extracellular vesicles comprises a diameter that is at least atleast about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm,about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000nm, or about 5,000 nm. In some embodiments, the heterogeneous populationof the extracellular vesicles comprises a diameter that is at least atmost about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm,about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000nm, or about 10,000 nm.

In some embodiments, the composition comprises a homogeneous populationof a plurality of extracellular vesicles. In some embodiments, thehomogeneous population of the extracellular vesicles comprises adiameter between about 10 nm to about 150 nm. In some embodiments, thehomogeneous population of the extracellular vesicles comprises adiameter between about 10 nm to about 30 nm, about 10 nm to about 40 nm,about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm toabout 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm,about 10 nm to about 100 nm, about 10 nm to about 110 nm, about 10 nm toabout 120 nm, about 10 nm to about 150 nm, about 30 nm to about 40 nm,about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm toabout 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm,about 30 nm to about 100 nm, about 30 nm to about 110 nm, about 30 nm toabout 120 nm, about 30 nm to about 150 nm, about 40 nm to about 50 nm,about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm toabout 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm,about 40 nm to about 110 nm, about 40 nm to about 120 nm, about 40 nm toabout 150 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm,about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm toabout 100 nm, about 50 nm to about 110 nm, about 50 nm to about 120 nm,about 50 nm to about 150 nm, about 60 nm to about 70 nm, about 60 nm toabout 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm,about 60 nm to about 110 nm, about 60 nm to about 120 nm, about 60 nm toabout 150 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm,about 70 nm to about 100 nm, about 70 nm to about 110 nm, about 70 nm toabout 120 nm, about 70 nm to about 150 nm, about 80 nm to about 90 nm,about 80 nm to about 100 nm, about 80 nm to about 110 nm, about 80 nm toabout 120 nm, about 80 nm to about 150 nm, about 90 nm to about 100 nm,about 90 nm to about 110 nm, about 90 nm to about 120 nm, about 90 nm toabout 150 nm, about 100 nm to about 110 nm, about 100 nm to about 120nm, about 100 nm to about 150 nm, about 110 nm to about 120 nm, about110 nm to about 150 nm, or about 120 nm to about 150 nm. In someembodiments, the homogeneous population of the extracellular vesiclescomprises a diameter between about 10 nm, about 30 nm, about 40 nm,about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about100 nm, about 110 nm, about 120 nm, or about 150 nm. In someembodiments, the homogeneous population of the extracellular vesiclescomprises a diameter between about at least about 10 nm, about 30 nm,about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about90 nm, about 100 nm, about 110 nm, or about 120 nm. In some embodiments,the homogeneous population of the extracellular vesicles comprises adiameter between about at most about 30 nm, about 40 nm, about 50 nm,about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about110 nm, about 120 nm, or about 150 nm.

Immune Checkpoint Moiety

Discredited herein, in some embodiments, are compositions comprisingextracellular vesicles comprising immune checkpoint moiety. In someembodiments, the extracellular vesicle comprises a plurality of immunecheckpoint moieties, where the immune check point moieties can be thesame or different. In some embodiments, the immune checkpoint moiety isencapsulated by the extracellular vesicle. In some embodiments, theimmune checkpoint moiety is expressed on the surface of theextracellular vesicle. In some embodiments, the immune checkpoint moietyis secreted by the extracellular vesicle. In some embodiments, theimmune checkpoint moiety is encapsulated by the extracellular vesicle.In some embodiments, the immune checkpoint moiety is expressed on thesurface of the extracellular vesicle; is secreted by the extracellularvesicle; is delivered by the extracellular to a target cell or a targetmicroenvironment; or a combination thereof. In some embodiments, theimmune checkpoint moiety comprises therapeutic properties for treating adisease or disorder. In some embodiments, the disease or disorder is anautoimmune disease. In some embodiments, the composition comprisingextracellular vesicle comprising the immune checkpoint moiety can beadministered to a subject to treat a disease or disorder.

In some embodiments, the immune checkpoint moiety comprises apolypeptide comprising a peptide sequence encoding VISTA, PD-L1, CTLA-4,PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H3, B7-H4(VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9,Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2,CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L,OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3,CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, or SIRPalpha (CD172a). In some embodiments, the immune checkpoint moietycomprises a peptide sequence encoding VISTA or a variation thereof or afragment thereof. In some embodiments, the immune checkpoint moietycomprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%,75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 1. In someembodiments, the immune checkpoint moiety comprises a peptide sequencethat is 100% identical to SEQ ID NO: 1 (Table 1). In some embodiments,the immune checkpoint moiety comprises a peptide sequence encoding PD-L1or a variation thereof or a fragment thereof. In some embodiments, theimmune checkpoint moiety comprises a peptide sequence that is at least50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical toSEQ ID NO: 2. In some embodiments, the immune checkpoint moietycomprises a peptide sequence that is 100% identical to SEQ ID NO: 2(Table 1). In some embodiments, the immune checkpoint moiety comprises apeptide sequence encoding CTLA-4 or a variation thereof or a fragmentthereof. In some embodiments, the immune checkpoint moiety comprises apeptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%.85%. 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments,the immune checkpoint moiety comprises a peptide sequence that is 100%identical to SEQ ID NO: 3 (Table 1).

TABLE 1 Peptide Sequence of VISTA, CTLA-4, PD-L1, and CTLA-4 SEQ. ID NOVISTA, Accession Number Q9H7M9 1MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVIPD-L1 (CD274), Accession Number Q9NZQ7 2MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET CTLA-4, Accession Number P16410 3MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATE VRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPP PYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

In some embodiments, the immune checkpoint moiety comprises aheterologous polynucleotide. In some embodiments, the heterologouspolynucleotide comprises mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA,snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, andshRNA. In some cases, the heterologous polynucleotide comprises mRNA. Insome embodiments, the heterologous polynucleotide encodes a nucleicsequence of VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3(CD276), B7-H2, B7-H3, B7-H4 (VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270,TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR(CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47,Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2(CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4(CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3(CD223), PECAM-1, CD44, or SIRP alpha (CD172a). In some embodiments, theimmune checkpoint moiety comprises a heterologous polynucleotideencoding VISTA. In some embodiments, the immune checkpoint moietycomprises a heterologous polynucleotide encoding PD-L1. In someembodiments, the immune checkpoint moiety comprises a heterologouspolynucleotide encoding CTLA-4.

In some embodiments, the immune checkpoint moiety comprises aheterologous polynucleotide encoding a cytokine. In some embodiments,the immune checkpoint moiety comprises a polypeptide comprising apeptide sequence of the cytokine. Exemplary cytokines that can beutilized as the immune check point moiety includes 4-1BBL, acylationstimulating protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6,CCL1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16,CCL17/TARC, CCL18, CCL19, CCL2, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC,CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4,CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L/CD178,Cerberus (protein), chemokines, CLCF1, CNTF, colony-stimulating factor,common b chain (CD131), common g chain (CD132), CX3CL1, CX3CR1, CXCL1,CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2,CXCL2/MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCR3, CXCR4,CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4,FAM19A5, Flt-3L, FMS-like tyrosine kinase 3 ligand, Foxp3, GATA-3,GcMAF, G-CSF, GITRL, GM-CSF, granulocyte colony-stimulating factor,granulocyte-macrophage colony-stimulating factor, hepatocyte growthfactor, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5/IFNaG, IFNA7,IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-α, IFN-β, IFN-γ, IFNω/IFNW1, IL-1,IL-10, IL-10 family, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-17 family, IL-17A-F, IL-18, IL-18BP, IL-19, IL-1A,IL-1B, IL-1F10, IL-1F3/IL-1RA, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9,IL-1-like, IL-1RA, IL-1RL2, IL-1α, IL-1β, IL-2, IL-20, IL-21, IL-22,IL-23, IL-24, IL-28A, IL-28B, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4,IL-5, IL-6, IL-6-like, IL-7, IL-8/CXCL8, IL-9, inflammasome,interferome, interferon, interferon beta-1a, interferon beta-1b,interferon gamma, interferon type I, interferon type II, interferon typeIII, interferons, interleukin, interleukin 1 receptor antagonist,Interleukin 8, IRF4, Leptin, leukemia inhibitory factor (LIF),leukocyte-promoting factor, LIGHT, LTA/TNFB, LT-β, lymphokine,lymphotoxin, lymphotoxin alpha, lymphotoxin beta, macrophagecolony-stimulating factor, macrophage inflammatory protein,macrophage-activating factor, M-CSF, MHC class III, miscellaneoushematopoietins, monokine, MSP, myokine, myonectin, nicotinamidephosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L,platelet factor 4, promegapoietin, RANKL, SCF, STAT3, STAT4, STATE,stromal cell-derived factor 1, TALL-1, TBX21, TGF-α, TGF-β, TGF-β1,TGF-β2, TGF-β3, TNF, TNF SF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-α, TNF-β, Tpo, TRAIL, TRANCE, TWEAK, vascularendothelial growth inhibitor, XCL1, or XCL2.

In some embodiments, the immune checkpoint moiety can be complexed withthe transmembrane moiety described herein. In some embodiments, theimmune checkpoint moiety can be non-covalently complexed with thetransmembrane moiety described herein. In some embodiments, the immunecheckpoint moiety can be covalently complexed with the transmembranemoiety described herein. In some embodiments, the immune checkpointmoiety can be expressed as part of a fusion protein comprising both theimmune checkpoint moiety and the transmembrane moiety. In someembodiments, the immune checkpoint moiety can be expressed as part of afusion protein comprising both the immune checkpoint moiety and afragment of the transmembrane moiety. In some embodiments, theN-terminus of the immune checkpoint moiety can be fused to thetransmembrane moiety. In some embodiments, the C-terminus of the immunecheckpoint moiety can be fused to the transmembrane moiety describedherein. In some embodiments, the immune checkpoint moiety can be fusedand flanked by the transmembrane moiety on both N and C-terminus of theimmune checkpoint moiety. For example, the immune checkpoint moiety canbe inserted into a transmembrane moiety as part of a fusion peptide,where the N-terminus of the fusion peptide comprises a fragment of thetransmembrane moiety, followed by the immune checkpoint moiety (or avariation there or a fragment thereof), and followed by the C-terminusof the fusion peptide comprising another fragment of the transmembranemoiety. In some embodiments, the immune checkpoint moiety comprises thefusion peptide, where the immune checkpoint moiety is fused to thetransmembrane moiety. In some embodiments, the immune checkpoint moietycomprises the immune checkpoint moiety complexed with the transmembranemoiety. In some embodiments, the immune checkpoint moiety comprises theimmune checkpoint moiety non-covalently complexed with the transmembranemoiety. In some embodiments, the immune checkpoint moiety comprises theimmune checkpoint moiety covalently complexed with the transmembranemoiety.

In some embodiments, the extracellular vesicle comprises a plurality ofthe immune checkpoint moiety described herein. In some embodiments, theplurality of the immune checkpoint moieties are encapsulated in theextracellular vesicle. In some embodiments, the extracellular vesicleencapsulates at least one, ten, 100, 500, 1,000, 5,000, 10,000, 50,000,100,000, 500,000, 1,000,000, 5,000,000, or more units of the immunecheckpoint moiety. In some embodiments, the extracellular vesicledelivers the encapsulated immune checkpoint to a target cell or a targetmicroenvironment.

In some embodiments, the extracellular vesicle secretes a plurality ofthe immune checkpoint moiety described herein. In some embodiments, theextracellular vesicle secretes at least one, ten, 100, 500, 1,000,5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or moreunits of the immune checkpoint moiety. In some embodiments, theextracellular vesicle secretes the immune checkpoint moiety to a targetcell or a target environment.

In some embodiments, the plurality of the immune checkpoint moiety isexpressed on the surface of the extracellular vesicle. In someembodiments, the plurality of the immune checkpoint moieties areexpressed as part of the fusion peptide comprising immune checkpointmoiety and transmembrane moiety. In some instances, the extracellularvesicle comprising the immune checkpoint moiety expressed on the surfaceof the extracellular vesicle contacts with a target cell or a targetenvironment.

In some embodiments, the number of units of the immune checkpoint moietythat can be expressed on the surface of the extracellular vesicle islimited by a theoretical maximum as determined by the ratio between: thedimensions of the extracellular vesicle; and the dimensions of theexpressed immune checkpoint moiety or the expressed fusion peptidecomprising the immune checkpoint moiety. In some embodiments, theplatforms and methods described herein can generate and select for anextracellular vesicle expressing a number of units of immune checkpointmoiety that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum of number ofunits of immune checkpoint moiety that can expressed on the surface ofthe extracellular vesicle. In some embodiments, the platforms andmethods described herein can generate and select for a homogenouspopulation of extracellular vesicles expressing a number of units ofimmune checkpoint moiety that is at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoreticalmaximum of number of units of immune checkpoint moiety that canexpressed on the surface of the extracellular vesicle. In someembodiments, the platforms and methods described herein can generate andselect for a homogenous population of extracellular vesicles expressinga number of units of immune checkpoint moiety on the surface of theextracellular vesicle that is at least 30% of the theoretical maximumnumber of units of immune checkpoint moiety that can be expressed on thesurface of the extracellular vesicle. In some embodiments, the platformsand methods described herein can generate and select for a homogenouspopulation of extracellular vesicles expressing a number of units ofimmune checkpoint moiety on the surface of the extracellular vesiclethat is at least 70% of the theoretical maximum number of units ofimmune checkpoint moiety that can be expressed on the surface of theextracellular vesicle. In some embodiments, the platforms and methodsdescribed herein can generate and select for a homogenous population ofextracellular vesicles expressing a number of units of immune checkpointmoiety on the surface of the extracellular vesicle that is at least 75%of the theoretical maximum number of units of immune checkpoint moietythat can be expressed on the surface of the extracellular vesicle. Insome embodiments, the platforms and methods described herein cangenerate and select for a homogenous population of extracellularvesicles expressing a number of units of immune checkpoint moiety on thesurface of the extracellular vesicle that is at least 80% of thetheoretical maximum number of units of immune checkpoint moiety that canbe expressed on the surface of the extracellular vesicle. In someembodiments, the platforms and methods described herein can generate andselect for a homogenous population of extracellular vesicles expressinga number of units of immune checkpoint moiety on the surface of theextracellular vesicle that is at least 85% of the theoretical maximumnumber of units of immune checkpoint moiety that can be expressed on thesurface of the extracellular vesicle. In some embodiments, the platformsand methods described herein can generate and select for a homogenouspopulation of extracellular vesicles expressing a number of units ofimmune checkpoint moiety on the surface of the extracellular vesiclethat is at least 90% of the theoretical maximum number of units ofimmune checkpoint moiety that can be expressed on the surface of theextracellular vesicle. In some embodiments, the platforms and methodsdescribed herein can generate and select for a homogenous population ofextracellular vesicles expressing a number of units of immune checkpointmoiety on the surface of the extracellular vesicle that is at least 95%of the theoretical maximum number of units of immune checkpoint moietythat can be expressed on the surface of the extracellular vesicle. Insome embodiments, the platforms and methods described herein cangenerate and select for a homogenous population of extracellularvesicles expressing a number of units of immune checkpoint moiety on thesurface of the extracellular vesicle that is at least 95% of thetheoretical maximum number of units of immune checkpoint moiety that canbe expressed on the surface of the extracellular vesicle.

In some embodiments, each extracellular vesicle expresses a number ofunits of immune checkpoint moiety on the surface of the extracellularvesicle, where the number can be about 5 units to about 1,000,000 units.In some embodiments, each extracellular vesicle expresses a number ofunits of immune checkpoint moiety on the surface of the extracellularvesicle, where the number can be about 5 units to about 10 units, about5 units to about 50 units, about 5 units to about 100 units, about 5units to about 500 units, about 5 units to about 1,000 units, about 5units to about 5,000 units, about 5 units to about 10,000 units, about 5units to about 50,000 units, about 5 units to about 100,000 units, about5 units to about 500,000 units, about 5 units to about 1,000,000 units,about 10 units to about 50 units, about 10 units to about 100 units,about 10 units to about 500 units, about 10 units to about 1,000 units,about 10 units to about 5,000 units, about 10 units to about 10,000units, about 10 units to about 50,000 units, about 10 units to about100,000 units, about 10 units to about 500,000 units, about 10 units toabout 1,000,000 units, about 50 units to about 100 units, about 50 unitsto about 500 units, about 50 units to about 1,000 units, about 50 unitsto about 5,000 units, about 50 units to about 10,000 units, about 50units to about 50,000 units, about 50 units to about 100,000 units,about 50 units to about 500,000 units, about 50 units to about 1,000,000units, about 100 units to about 500 units, about 100 units to about1,000 units, about 100 units to about 5,000 units, about 100 units toabout 10,000 units, about 100 units to about 50,000 units, about 100units to about 100,000 units, about 100 units to about 500,000 units,about 100 units to about 1,000,000 units, about 500 units to about 1,000units, about 500 units to about 5,000 units, about 500 units to about10,000 units, about 500 units to about 50,000 units, about 500 units toabout 100,000 units, about 500 units to about 500,000 units, about 500units to about 1,000,000 units, about 1,000 units to about 5,000 units,about 1,000 units to about 10,000 units, about 1,000 units to about50,000 units, about 1,000 units to about 100,000 units, about 1,000units to about 500,000 units, about 1,000 units to about 1,000,000units, about 5,000 units to about 10,000 units, about 5,000 units toabout 50,000 units, about 5,000 units to about 100,000 units, about5,000 units to about 500,000 units, about 5,000 units to about 1,000,000units, about 10,000 units to about 50,000 units, about 10,000 units toabout 100,000 units, about 10,000 units to about 500,000 units, about10,000 units to about 1,000,000 units, about 50,000 units to about100,000 units, about 50,000 units to about 500,000 units, about 50,000units to about 1,000,000 units, about 100,000 units to about 500,000units, about 100,000 units to about 1,000,000 units, or about 500,000units to about 1,000,000 units. In some embodiments, each extracellularvesicle expresses a number of units of immune checkpoint moiety on thesurface of the extracellular vesicle, where the number can be about 5units, about 10 units, about 50 units, about 100 units, about 500 units,about 1,000 units, about 5,000 units, about 10,000 units, about 50,000units, about 100,000 units, about 500,000 units, or about 1,000,000units. In some embodiments, each extracellular vesicle expresses anumber of units of immune checkpoint moiety on the surface of theextracellular vesicle, where the number can be at least about 5 units,about 10 units, about 50 units, about 100 units, about 500 units, about1,000 units, about 5,000 units, about 10,000 units, about 50,000 units,about 100,000 units, or about 500,000 units. In some embodiments, eachextracellular vesicle expresses a number of units of immune checkpointmoiety on the surface of the extracellular vesicle, where the number canbe at most about 10 units, about 50 units, about 100 units, about 500units, about 1,000 units, about 5,000 units, about 10,000 units, about50,000 units, about 100,000 units, about 500,000 units, or about1,000,000 units.

In some embodiments, the composition described herein comprises aheterogenous population of extracellular vesicles expressing any numberof units of the immune checkpoint moiety described herein on the surfaceof the extracellular vesicles. In some embodiments, the compositiondescribed herein comprises a homogeneous population of extracellularvesicles expressing a range of number of units of the immune checkpointmoiety on the surface of the extracellular vesicles. In someembodiments, the homogeneous population of extracellular vesiclesexpresses a number of units of the immune checkpoint moiety that is atleast about 5 units to about 1,000,000 units. In some embodiments, thehomogeneous population of extracellular vesicles expresses a number ofunits of the immune checkpoint moiety that is at least about 5 units toabout 10 units, about 5 units to about 50 units, about 5 units to about100 units, about 5 units to about 500 units, about 5 units to about1,000 units, about 5 units to about 5,000 units, about 5 units to about10,000 units, about 5 units to about 50,000 units, about 5 units toabout 100,000 units, about 5 units to about 500,000 units, about 5 unitsto about 1,000,000 units, about 10 units to about 50 units, about 10units to about 100 units, about 10 units to about 500 units, about 10units to about 1,000 units, about 10 units to about 5,000 units, about10 units to about 10,000 units, about 10 units to about 50,000 units,about 10 units to about 100,000 units, about 10 units to about 500,000units, about 10 units to about 1,000,000 units, about 50 units to about100 units, about 50 units to about 500 units, about 50 units to about1,000 units, about 50 units to about 5,000 units, about 50 units toabout 10,000 units, about 50 units to about 50,000 units, about 50 unitsto about 100,000 units, about 50 units to about 500,000 units, about 50units to about 1,000,000 units, about 100 units to about 500 units,about 100 units to about 1,000 units, about 100 units to about 5,000units, about 100 units to about 10,000 units, about 100 units to about50,000 units, about 100 units to about 100,000 units, about 100 units toabout 500,000 units, about 100 units to about 1,000,000 units, about 500units to about 1,000 units, about 500 units to about 5,000 units, about500 units to about 10,000 units, about 500 units to about 50,000 units,about 500 units to about 100,000 units, about 500 units to about 500,000units, about 500 units to about 1,000,000 units, about 1,000 units toabout 5,000 units, about 1,000 units to about 10,000 units, about 1,000units to about 50,000 units, about 1,000 units to about 100,000 units,about 1,000 units to about 500,000 units, about 1,000 units to about1,000,000 units, about 5,000 units to about 10,000 units, about 5,000units to about 50,000 units, about 5,000 units to about 100,000 units,about 5,000 units to about 500,000 units, about 5,000 units to about1,000,000 units, about 10,000 units to about 50,000 units, about 10,000units to about 100,000 units, about 10,000 units to about 500,000 units,about 10,000 units to about 1,000,000 units, about 50,000 units to about100,000 units, about 50,000 units to about 500,000 units, about 50,000units to about 1,000,000 units, about 100,000 units to about 500,000units, about 100,000 units to about 1,000,000 units, or about 500,000units to about 1,000,000 units. In some embodiments, the homogeneouspopulation of extracellular vesicles expresses a number of units of theimmune checkpoint moiety that is at least about 5 units, about 10 units,about 50 units, about 100 units, about 500 units, about 1,000 units,about 5,000 units, about 10,000 units, about 50,000 units, about 100,000units, about 500,000 units, or about 1,000,000 units. In someembodiments, the homogeneous population of extracellular vesiclesexpresses a number of units of the immune checkpoint moiety that is atleast at least about 5 units, about 10 units, about 50 units, about 100units, about 500 units, about 1,000 units, about 5,000 units, about10,000 units, about 50,000 units, about 100,000 units, or about 500,000units. In some embodiments, the homogeneous population of extracellularvesicles expresses a number of units of the immune checkpoint moietythat is at least at most about 10 units, about 50 units, about 100units, about 500 units, about 1,000 units, about 5,000 units, about10,000 units, about 50,000 units, about 100,000 units, about 500,000units, or about 1,000,000 units.

In some embodiments, the homogeneous population of extracellularvesicles expresses a number of units of the immune checkpoint moietythat is at most about 5 units to about 1,000,000 units. In someembodiments, the homogeneous population of extracellular vesiclesexpresses a number of units of the immune checkpoint moiety that is atmost about 5 units to about 10 units, about 5 units to about 50 units,about 5 units to about 100 units, about 5 units to about 500 units,about 5 units to about 1,000 units, about 5 units to about 5,000 units,about 5 units to about 10,000 units, about 5 units to about 50,000units, about 5 units to about 100,000 units, about 5 units to about500,000 units, about 5 units to about 1,000,000 units, about 10 units toabout 50 units, about 10 units to about 100 units, about 10 units toabout 500 units, about 10 units to about 1,000 units, about 10 units toabout 5,000 units, about 10 units to about 10,000 units, about 10 unitsto about 50,000 units, about 10 units to about 100,000 units, about 10units to about 500,000 units, about 10 units to about 1,000,000 units,about 50 units to about 100 units, about 50 units to about 500 units,about 50 units to about 1,000 units, about 50 units to about 5,000units, about 50 units to about 10,000 units, about 50 units to about50,000 units, about 50 units to about 100,000 units, about 50 units toabout 500,000 units, about 50 units to about 1,000,000 units, about 100units to about 500 units, about 100 units to about 1,000 units, about100 units to about 5,000 units, about 100 units to about 10,000 units,about 100 units to about 50,000 units, about 100 units to about 100,000units, about 100 units to about 500,000 units, about 100 units to about1,000,000 units, about 500 units to about 1,000 units, about 500 unitsto about 5,000 units, about 500 units to about 10,000 units, about 500units to about 50,000 units, about 500 units to about 100,000 units,about 500 units to about 500,000 units, about 500 units to about1,000,000 units, about 1,000 units to about 5,000 units, about 1,000units to about 10,000 units, about 1,000 units to about 50,000 units,about 1,000 units to about 100,000 units, about 1,000 units to about500,000 units, about 1,000 units to about 1,000,000 units, about 5,000units to about 10,000 units, about 5,000 units to about 50,000 units,about 5,000 units to about 100,000 units, about 5,000 units to about500,000 units, about 5,000 units to about 1,000,000 units, about 10,000units to about 50,000 units, about 10,000 units to about 100,000 units,about 10,000 units to about 500,000 units, about 10,000 units to about1,000,000 units, about 50,000 units to about 100,000 units, about 50,000units to about 500,000 units, about 50,000 units to about 1,000,000units, about 100,000 units to about 500,000 units, about 100,000 unitsto about 1,000,000 units, or about 500,000 units to about 1,000,000units. In some embodiments, the homogeneous population of extracellularvesicles expresses a number of units of the immune checkpoint moietythat is at most about 5 units, about 10 units, about 50 units, about 100units, about 500 units, about 1,000 units, about 5,000 units, about10,000 units, about 50,000 units, about 100,000 units, about 500,000units, or about 1,000,000 units. In some embodiments, the homogeneouspopulation of extracellular vesicles expresses a number of units of theimmune checkpoint moiety that is at most at least about 5 units, about10 units, about 50 units, about 100 units, about 500 units, about 1,000units, about 5,000 units, about 10,000 units, about 50,000 units, about100,000 units, or about 500,000 units. In some embodiments, thehomogeneous population of extracellular vesicles expresses a number ofunits of the immune checkpoint moiety that is at most at most about 10units, about 50 units, about 100 units, about 500 units, about 1,000units, about 5,000 units, about 10,000 units, about 50,000 units, about100,000 units, about 500,000 units, or about 1,000,000 units.

In some embodiments, the homogeneous population of extracellularvesicles expresses a number of units of the immune checkpoint moietythat is between about 1,000 units to about 10,000 units. In someembodiments, the homogeneous population of extracellular vesiclesexpresses a number of units of the immune checkpoint moiety that isbetween about 1,000 units to about 1,500 units, about 1,000 units toabout 2,000 units, about 1,000 units to about 2,500 units, about 1,000units to about 3,000 units, about 1,000 units to about 4,000 units,about 1,000 units to about 5,000 units, about 1,000 units to about 6,000units, about 1,000 units to about 7,000 units, about 1,000 units toabout 8,000 units, about 1,000 units to about 9,000 units, about 1,000units to about 10,000 units, about 1,500 units to about 2,000 units,about 1,500 units to about 2,500 units, about 1,500 units to about 3,000units, about 1,500 units to about 4,000 units, about 1,500 units toabout 5,000 units, about 1,500 units to about 6,000 units, about 1,500units to about 7,000 units, about 1,500 units to about 8,000 units,about 1,500 units to about 9,000 units, about 1,500 units to about10,000 units, about 2,000 units to about 2,500 units, about 2,000 unitsto about 3,000 units, about 2,000 units to about 4,000 units, about2,000 units to about 5,000 units, about 2,000 units to about 6,000units, about 2,000 units to about 7,000 units, about 2,000 units toabout 8,000 units, about 2,000 units to about 9,000 units, about 2,000units to about 10,000 units, about 2,500 units to about 3,000 units,about 2,500 units to about 4,000 units, about 2,500 units to about 5,000units, about 2,500 units to about 6,000 units, about 2,500 units toabout 7,000 units, about 2,500 units to about 8,000 units, about 2,500units to about 9,000 units, about 2,500 units to about 10,000 units,about 3,000 units to about 4,000 units, about 3,000 units to about 5,000units, about 3,000 units to about 6,000 units, about 3,000 units toabout 7,000 units, about 3,000 units to about 8,000 units, about 3,000units to about 9,000 units, about 3,000 units to about 10,000 units,about 4,000 units to about 5,000 units, about 4,000 units to about 6,000units, about 4,000 units to about 7,000 units, about 4,000 units toabout 8,000 units, about 4,000 units to about 9,000 units, about 4,000units to about 10,000 units, about 5,000 units to about 6,000 units,about 5,000 units to about 7,000 units, about 5,000 units to about 8,000units, about 5,000 units to about 9,000 units, about 5,000 units toabout 10,000 units, about 6,000 units to about 7,000 units, about 6,000units to about 8,000 units, about 6,000 units to about 9,000 units,about 6,000 units to about 10,000 units, about 7,000 units to about8,000 units, about 7,000 units to about 9,000 units, about 7,000 unitsto about 10,000 units, about 8,000 units to about 9,000 units, about8,000 units to about 10,000 units, or about 9,000 units to about 10,000units. In some embodiments, the homogeneous population of extracellularvesicles expresses a number of units of the immune checkpoint moietythat is between about 1,000 units, about 1,500 units, about 2,000 units,about 2,500 units, about 3,000 units, about 4,000 units, about 5,000units, about 6,000 units, about 7,000 units, about 8,000 units, about9,000 units, or about 10,000 units. In some embodiments, the homogeneouspopulation of extracellular vesicles expresses a number of units of theimmune checkpoint moiety that is between at least about 1,000 units,about 1,500 units, about 2,000 units, about 2,500 units, about 3,000units, about 4,000 units, about 5,000 units, about 6,000 units, about7,000 units, about 8,000 units, or about 9,000 units. In someembodiments, the homogeneous population of extracellular vesiclesexpresses a number of units of the immune checkpoint moiety that isbetween at most about 1,500 units, about 2,000 units, about 2,500 units,about 3,000 units, about 4,000 units, about 5,000 units, about 6,000units, about 7,000 units, about 8,000 units, about 9,000 units, or about10,000 units.

Transmembrane Moiety

Discredited herein, in some embodiments, are compositions comprisingextracellular vesicles comprising transmembrane moiety. In someembodiments, the transmembrane moiety comprises a full length protein ora variation thereof or a fragment thereof. In some embodiments, thetransmembrane is selected from a group consisting of: 14-3-3 proteinzeta/delta, 4-3-3 protein epsilon, 78 kDa glucose-regulated protein,acetylcholinesterase/AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA),ADAM10, alkaline phosphatase, alpha-enolase, alpha-synuclein,aminopeptidase N, amyloid beta A4/APP, annexin 5A, annexin A2, AP-1,ATF3, ATP citrate lyase, ATPase, beta actin (ACTB), beta-amyloid 42,caveolin-1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24,CD26/DPP4, CD29/ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49,CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudin,claudin-1 cofilin-1, complement-binding proteins CD55 and CD59,cytosolic heat shock protein 90 alpha, cytosolic heat shock protein 90beta, EBV LMP1, EBV LMP2A, EF-1alpha-1, EF2, EFGR EGFR VIII,emmprin/CD147, enolase 1 alpha (ENO1), EPCAM, ERBB2, tetraspanins (CD9,CD63 and CD81), fatty acid synthase, fetuin-A, flotillin-1, flotillin-2,fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5′nucleotidase,GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 andHSP90), heparan sulfate proteoglycans, heparinase, heterotrimeric Gproteins, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax,huntingtin, ICAM1, integrins, lactadherin, LAMP1/2, LAMP2b, leucine-richreceptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associatedmembrane glycoprotein 1, lysosome-associated membrane glycoprotein 2,MEW class I, MEW class II, MUC1, multidrug resistance-associatedprotein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP/Alix,PECAM1, phosphoglycerate kinase, placental prion proteins,prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a,Rab-5b, Rab-5c, Rab-7, Rap 1B, resistin, sonic hedgehog (SHH),surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor(TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8,tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein,TYRP-2, vacuolar-sorting protein 35, or zeta polypeptide (YWHAZ). Insome embodiments, the transmembrane moiety comprises lactadherin. Insome embodiments, the transmembrane moiety comprises C1C2 domain oflactadherin. In some embodiments, the transmembrane moiety comprisesLAMP2. In some embodiments, the transmembrane moiety comprises LAMP-likedomain 1 of LAMP2. In some embodiments, the transmembrane moietycomprises LAMP2b. In some embodiments, the transmembrane moietycomprises a peptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%.85%. 90%, 95%, or 99% identical to SEQ ID NO: 4 (Table 2). In someembodiments, the transmembrane moiety comprises a peptide that is 100%identical to SEQ ID NO: 4. In some embodiments, the transmembrane moietycomprises glycosylphosphatidylinositol (GPI) protein. In someembodiments, the transmembrane moiety comprises glycan portion of GPI.In some embodiments, the transmembrane moiety comprises lipid portion ofGPI. In some cases, the transmembrane moiety comprises CD63. In someembodiments, the transmembrane moiety comprises a peptide is at least50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical toSEQ ID NO: 5 (Table 2). In some embodiments, the transmembrane moietycomprises a peptide that is 100% identical to SEQ ID NO: 5. In someembodiments, the transmembrane moiety comprises a modified CD63. In someembodiments, the modified CD63 comprises at least one CD63 transmembranedomain. The transmembrane domain can be transmembrane domain 1 (TM1) ofCD63, transmembrane domain 2 (TM2) of CD63, transmembrane domain 3 (TM3)of CD63, transmembrane domain 4 (TM4) of CD63, or any combinationthereof. In some embodiments, the modified CD63 comprises one, two,three, four, five, six, seven, eight, nine, ten or more of transmembranedomains of CD63. In some embodiments, the modified CD63 comprises onetransmembrane domain. In some embodiments, the modified CD63 comprisestwo transmembrane domains. In some embodiments, the modified CD63comprises three transmembrane domains. In some embodiments, the modifiedCD63 comprises four transmembrane domains. In some embodiments, themodified CD63 comprises five transmembrane domains. In some embodiments,the modified CD63 comprises six transmembrane domains. In someembodiments, the modified CD63 comprises seven transmembrane domains. Insome embodiments, the modified CD63 comprises eight transmembranedomains. In some embodiments, the modified CD63 comprises ninetransmembrane domains. In some embodiments, the modified CD63 comprisesten transmembrane domains. In some embodiments, the modified CD63 can bea truncated CD63, where at least one transmembrane domain is removed. Insome embodiments, the modified CD63 comprises both truncation of CD63and additional of at least one transmembrane domain of CD63. Forexample, a modified CD63 can be truncated at N-terminus to remove TM1and further comprises additional TM3 and TM4, resulting in a modifiedCD63 comprising, in order of transmembrane domains of CD63, TM2, TM3,TM4, TM3, and TM4.

TABLE 2 Peptide Sequences of Lamp2b and CD63 SEQ ID NOLamp2b, Acccesion Number P13473-2 4MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQDCSADDDNFLVPIAVGAALAGVLILVLLAYFIGLKHHHAG YEQFCD63, Accession Number A0A024RB05 5MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVII AVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNN FRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCG INFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM

In some embodiments, the modified CD63 comprises inserting orsubstituting the non-transmembrane domains (either cytoplasmic loop orextracellular loop) of the modified CD63. In some embodiments, themodified CD63 comprises a cytoplasmic loop inserted or substituted withthe immune checkpoint moiety. In some embodiments, the modified CD63comprises an extracellular loop inserted or substituted with the immunecheckpoint moiety. For example, a polypeptide comprising the immunecheckpoint moiety can be inserted into the extracellular loop of themodified CD63. Alternatively, polypeptide comprising the immunecheckpoint moiety can substitute a fragment of the extracellular loop ofthe modified CD63. In some embodiments, the immune checkpoint moiety canbe fused to a truncated version of the modified CD63. In someembodiments, the immune checkpoint moiety can be fused to the N-terminusof the modified CD63, where the modified CD63 is truncated at theN-terminus to remove at least one of the transmembrane domains and/or atleast one of the non-transmembrane domains. In some embodiments, theimmune checkpoint moiety can be fused to the N-terminus of the modifiedCD63, where the modified CD63 is truncated at the N-terminus to removeat least one of the transmembrane domains and/or at least one of thenon-transmembrane domains.

In some embodiments, the immune checkpoint moiety can be fused to thetransmembrane moiety such as CD63 via a linker. In some embodiments, thelinker is a linker peptide. The linker peptides not only serves toconnect the moieties, but in some cases also provides many otherfunctions, such as maintaining cooperative inter-domain interactions orpreserving biological activity (Gokhale R S, Khosla C. Role of linkersin communication between protein modules. Curr Opin Chem Biol. 2000; 4:22-27; Ikebe M, Kambara T, Stafford W F, Sata M, Katayama E, Ikebe R. Ahinge at the central helix of the regulatory light chain of myosin iscritical for phosphorylation-dependent regulation of smooth musclemyosin motor activity. J Biol Chem. 1998; 273: 17702-17707; and Chen XY, Zaro J, and Shen W C. Fusion protein linkers: property, design andfunctionality. Adv Drug Deliv Rev 2014; 65, 1357-1369 are incorporatedherein). The linker peptides can be grouped into small, medium, andlarge linkers with average length of less than or up to 4.5±0.7,9.1±2.4, and 21.0±7.6 residues or greater, respectively, althoughexamples anywhere within the set defined by these three ranges are alsocontemplated. In some embodiments, the linker peptide comprises 5 to 200amino acids. In other embodiments, the linker peptide comprises 5 to 25amino acids. In some embodiments, the linker peptide is a cleavable(e.g., a linker peptide comprising a peptide sequence that can berecognized and cleaved by Tev protease).

SEQ ID NOS: 6-17 illustrate various arrangements of the immunecheckpoint moiety PD-L1 fusing to the transmembrane moiety CD63 (Table3).

TABLE 3Peptide Sequence of Fusion of Immune Checkpoint Moiety and TransmembraneMoiety SEQ ID Immune Checkpoint Moiety and Transmembrance Moiety FusionNO Peptide Comment 6MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVG X is anVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEGGGGS[X]extracellular domain of an Immune Checkpoint Inhibitor 7MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 8MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV X is anFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEGGGGSGGGGSextracellular GGGGS[X] domain of an Immune Checkpoint Inhibitor 9MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEGGGGSGGGGSGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 10MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV whereinFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFR X is anQQGGGGSX extracellular domain of an Immune Checkpoint Inhibitor 11MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRQQGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 12MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV X is anFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRextracellular QQGGGGSGGGGSGGGGS[X] domain of an Immune CheckpointInhibitor 13MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRQQGGGGSGGGGSGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 14MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV X is anFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRextracellular QQMENYPKNNHTGGGGSX domain of an Immune CheckpointInhibitor 15MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRQQMENYPKNNHTGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 16MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV X is anFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRextracellular QQMENYPKNNHTGGGGSGGGGSGGGGS[X] domain of an 17MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGV 0FLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEVMSEFNNNFRQQMENYPKNNHTGGGGSGGGGSGGGGSFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 6. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 6. In some embodiments,the expressed polypeptide corresponding to SEQ ID NO: 6 is partiallyembedded in the membrane of the extracellular vesicle and is partiallyexpressed on the surface of the extracellular vesicle.

7 In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 7. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 7. In some embodiments,the expressed polypeptide corresponding to SEQ ID NO: 7 is partiallyembedded in the membrane of the extracellular vesicle and is partiallyexpressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 8. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 8. In some embodiments,the expressed polypeptide corresponding to SEQ ID NO: 8 is partiallyembedded in the membrane of the extracellular vesicle and is partiallyexpressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 9. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 9. In some embodiments,the expressed polypeptide corresponding to SEQ ID NO: 9 is partiallyembedded in the membrane of the extracellular vesicle and is partiallyexpressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 10. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 10. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 10 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 11. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 11. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 11 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 12. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 12. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 12 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 13. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 13. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 13 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 14. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 14. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 14 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 15. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 15. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 15 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 16. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 16. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 16 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the extracellular vesicle described hereinexpresses a polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 17. In someembodiments, the extracellular vesicle described herein expresses apolypeptide that is 100% identical to SEQ ID NO: 17. In someembodiments, the expressed polypeptide corresponding to SEQ ID NO: 17 ispartially embedded in the membrane of the extracellular vesicle and ispartially expressed on the surface of the extracellular vesicle.

In some embodiments, the transmembrane moiety can be complexed with theimmune checkpoint moiety described herein. In some embodiments, thetransmembrane moiety can be non-covalently complex with the immunecheckpoint moiety described herein. In some embodiments, thetransmembrane moiety can be covalently complexed with the immunecheckpoint moiety described herein. In some embodiments, thetransmembrane moiety can be fused to the immune checkpoint moietydescribed herein at the N-terminus of the transmembrane moiety. In someembodiments, the transmembrane moiety can be fused to the immunecheckpoint moiety described herein at the C-terminus of thetransmembrane moiety.

In some embodiments, the immune checkpoint moiety comprises therapeuticproperties for treating a disease or a disorder. In some embodiments,the immune checkpoint moiety comprises therapeutic properties fortreating an autoimmune disease described herein. In some embodiments,the immune checkpoint moiety targets and modulates activities of immunecells. In some embodiments, the immune cells can be T cell, includingCytotoxic T cell, Natural Killer T cell, Regulatory T cell, and T helpercells.

Targeting Moiety

Described herein, in some embodiments, are extracellular vesiclescomprising targeting moiety. In some embodiments, the targeting moietycan be expressed on the surface of the extracellular vesicle. In someembodiments, the targeting moiety can be secreted by the extracellularvesicle. The extracellular vehicles comprising the targeting moietylocalizes at the target cell or target environment is at least 2 fold, 5fold, 10 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1,000 fold, 5,000fold, or 10,000 fold higher compared to localization of an extracellularvesicle lacking the targeting moiety. In some embodiments, the targetingmoiety comprises EBV glycoprotein 350, which targets CD19+ B cells. Insome embodiments, the targeting moiety comprises Lamp2b, which targetsacetylcholine receptors on neurons. In some embodiments, the targetingmoiety comprises C1C2 domain of lactadherin, which target immune cellsor blood cells, In some embodiments, the targeting moiety comprisesPDGFR, which targets EGFR or cells expressing EGFR. In some embodiments,the targeting moiety comprises GPI-anchored membrane proteins.

In some embodiments, the targeting moiety can target a cell surfaceprotein or a protein secreted by the target cell. Non-limiting examplesof the cell surface or secreted proteins include any one of thechemokines described herein.

Pharmaceutical Compositions

Described herein are pharmaceutical compositions comprising thecompositions described herein. In some embodiments, the pharmaceuticalcomposition comprises both the composition comprising the extracellularvesicle and the cells that secret the extracellular vesicles. Apharmaceutical composition, as used herein, refers to a mixture of atherapeutic agent comprising extracellular vesicle, with other chemicalcomponents (i.e., pharmaceutically acceptable inactive ingredients),such as carriers, excipients, binders, filling agents, suspendingagents, flavoring agents, sweetening agents, disintegrating agents,dispersing agents, surfactants, lubricants, colorants, diluents,solubilizers, moistening agents, plasticizers, stabilizers, penetrationenhancers, wetting agents, anti-foaming agents, antioxidants,preservatives, or one or more combination thereof. Optionally, thecompositions include two or more therapeutic agent (e.g., one or moretherapeutic agents and one or more additional agents) as discussedherein. In practicing the methods of treatment or use provided herein,therapeutically effective amounts of therapeutic agents described hereinare administered in a pharmaceutical composition to a mammal having adisease, disorder, or condition to be treated, e.g., an autoimmunedisease. In some embodiments, the mammal is a human. A therapeuticallyeffective amount can vary widely depending on the severity of thedisease, the age and relative health of the subject, the potency of thetherapeutic agent used and other factors. The therapeutic agents can beused singly or in combination with one or more therapeutic agents ascomponents of mixtures.

The pharmaceutical formulations described herein are administered to asubject by appropriate administration routes, including but not limitedto, intravenous, intraarterial, oral, parenteral, buccal, topical,transdermal, rectal, intramuscular, subcutaneous, intraosseous,transmucosal, inhalation, or intraperitoneal administration routes. Thepharmaceutical formulations described herein include, but are notlimited to, aqueous liquid dispersions, self-emulsifying dispersions,solid solutions, liposomal dispersions, aerosols, solid dosage forms,powders, immediate release formulations, controlled releaseformulations, fast melt formulations, tablets, capsules, pills, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate andcontrolled release formulations.

Pharmaceutical compositions including a therapeutic agent aremanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The pharmaceutical compositions may include at least a therapeutic agentas an active ingredient in free-acid or free-base form, or in apharmaceutically acceptable salt form. In addition, the methods andpharmaceutical compositions described herein include the use of N-oxides(if appropriate), crystalline forms, amorphous phases, as well as activemetabolites of these compounds having the same type of activity. In someembodiments, therapeutic agents exist in unsolvated form or in solvatedforms with pharmaceutically acceptable solvents such as water, ethanol,and the like. The solvated forms of the therapeutic agents are alsoconsidered to be disclosed herein.

In some embodiments, a therapeutic agent exists as a tautomer. Alltautomers are included within the scope of the agents presented herein.As such, it is to be understood that a therapeutic agent or a saltthereof may exhibit the phenomenon of tautomerism whereby two chemicalcompounds that are capable of facile interconversion by exchanging ahydrogen atom between two atoms, to either of which it forms a covalentbond. Since the tautomeric compounds exist in mobile equilibrium witheach other they may be regarded as different isomeric forms of the samecompound.

In some embodiments, a therapeutic agent exists as an enantiomer,diastereomer, or other steroisomeric form. The agents disclosed hereininclude all enantiomeric, diastereomeric, and epimeric forms as well asmixtures thereof.

In some embodiments, therapeutic agents described herein may be preparedas prodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a therapeutic agent described herein,which is administered as an ester (the “prodrug”) to facilitatetransmittal across a cell membrane where water solubility is detrimentalto mobility but which then is metabolically hydrolyzed to the carboxylicacid, the active entity, once inside the cell where water-solubility isbeneficial. A further example of a prodrug might be a short peptide(polyaminoacid) bonded to an acid group where the peptide is metabolizedto reveal the active moiety. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the therapeuticagent. In certain embodiments, a prodrug is enzymatically metabolized byone or more steps or processes to the biologically, pharmaceutically ortherapeutically active form of the therapeutic agent.

Prodrug forms of the therapeutic agents, wherein the prodrug ismetabolized in vivo to produce an agent as set forth herein are includedwithin the scope of the claims. Prodrug forms of the herein describedtherapeutic agents, wherein the prodrug is metabolized in vivo toproduce an agent as set forth herein are included within the scope ofthe claims. In some cases, some of the therapeutic agents describedherein may be a prodrug for another derivative or active compound. Insome embodiments described herein, hydrazones are metabolized in vivo toproduce a therapeutic agent.

In certain embodiments, compositions provided herein include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

In some embodiments, formulations described herein benefit fromantioxidants, metal chelating agents, thiol containing compounds andother general stabilizing agents. Examples of such stabilizing agents,include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

The pharmaceutical compositions described herein are formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions,solid oral dosage forms, aerosols, controlled release formulations, fastmelt formulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations. In one aspect, a therapeutic agentas discussed herein, e.g., therapeutic agent is formulated into apharmaceutical composition suitable for intramuscular, subcutaneous, orintravenous injection. In one aspect, formulations suitable forintramuscular, subcutaneous, or intravenous injection includephysiologically acceptable sterile aqueous or non-aqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and non-aqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propyleneglycol,polyethylene-glycol, glycerol, cremophor and the like), suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants. In some embodiments, formulationssuitable for subcutaneous injection also contain additives such aspreserving, wetting, emulsifying, and dispensing agents. Prevention ofthe growth of microorganisms can be ensured by various antibacterial andantifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid,and the like. In some cases it is desirable to include isotonic agents,such as sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections or drips or infusions, a therapeutic agentdescribed herein is formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart. For other parenteral injections, appropriate formulations includeaqueous or nonaqueous solutions, preferably with physiologicallycompatible buffers or excipients. Such excipients are known.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. In one aspect, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

For administration by inhalation, a therapeutic agent is formulated foruse as an aerosol, a mist or a powder. Pharmaceutical compositionsdescribed herein are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the therapeutic agent described herein and a suitable powder basesuch as lactose or starch.

Representative intranasal formulations are described in, for example,U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Formulations thatinclude a therapeutic agent are prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, fluorocarbons,and/or other solubilizing or dispersing agents known in the art. See,for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are known to those skilled inthe preparation of nasal dosage forms and some of these can be found inREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005. Thechoice of suitable carriers is dependent upon the exact nature of thenasal dosage form desired, e.g., solutions, suspensions, ointments, orgels. Nasal dosage forms generally contain large amounts of water inaddition to the active ingredient. Minor amounts of other ingredientssuch as pH adjusters, emulsifiers or dispersing agents, preservatives,surfactants, gelling agents, or buffering and other stabilizing andsolubilizing agents are optionally present. Preferably, the nasal dosageform should be isotonic with nasal secretions.

Pharmaceutical preparations for oral use are obtained by mixing one ormore solid excipient with one or more of the therapeutic agentsdescribed herein, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude, for example, fillers such as sugars, including lactose,sucrose, mannitol, or sorbitol; cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents are added, such as the cross linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate. In some embodiments, dyestuffs orpigments are added to the tablets or dragee coatings for identificationor to characterize different combinations of active therapeutic agentdoses.

In some embodiments, pharmaceutical formulations of a therapeutic agentare in the form of a capsules, including push fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push fit capsules containthe active ingredients in admixture with filler such as lactose, binderssuch as starches, and/or lubricants such as talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active therapeuticagent is dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In some embodiments,stabilizers are added. A capsule may be prepared, for example, byplacing the bulk blend of the formulation of the therapeutic agentinside of a capsule. In some embodiments, the formulations (non-aqueoussuspensions and solutions) are placed in a soft gelatin capsule. Inother embodiments, the formulations are placed in standard gelatincapsules or non-gelatin capsules such as capsules comprising HPMC. Inother embodiments, the formulation is placed in a sprinkle capsule,wherein the capsule is swallowed whole or the capsule is opened and thecontents sprinkled on food prior to eating.

All formulations for oral administration are in dosages suitable forsuch administration. In one aspect, solid oral dosage forms are preparedby mixing a therapeutic agent with one or more of the following:antioxidants, flavoring agents, and carrier materials such as binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, and diluents. Insome embodiments, the solid dosage forms disclosed herein are in theform of a tablet, (including a suspension tablet, a fast-melt tablet, abite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder, a capsule, soliddispersion, solid solution, bioerodible dosage form, controlled releaseformulations, pulsatile release dosage forms, multiparticulate dosageforms, beads, pellets, granules. In other embodiments, thepharmaceutical formulation is in the form of a powder. Compressedtablets are solid dosage forms prepared by compacting the bulk blend ofthe formulations described above. In various embodiments, tablets willinclude one or more flavoring agents. In other embodiments, the tabletswill include a film surrounding the final compressed tablet. In someembodiments, the film coating can provide a delayed release of atherapeutic agent from the formulation. In other embodiments, the filmcoating aids in patient compliance (e.g., Opadry® coatings or sugarcoating). Film coatings including Opadry® typically range from about 1%to about 3% of the tablet weight. In some embodiments, solid dosageforms, e.g., tablets, effervescent tablets, and capsules, are preparedby mixing particles of a therapeutic agent with one or morepharmaceutical excipients to form a bulk blend composition. The bulkblend is readily subdivided into equally effective unit dosage forms,such as tablets, pills, and capsules. In some embodiments, theindividual unit dosages include film coatings. These formulations aremanufactured by conventional formulation techniques.

In another aspect, dosage forms include microencapsulated formulations.In some embodiments, one or more other compatible materials are presentin the microencapsulation material. Exemplary materials include, but arenot limited to, pH modifiers, erosion facilitators, anti-foaming agents,antioxidants, flavoring agents, and carrier materials such as binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, and diluents.Exemplary useful microencapsulation materials include, but are notlimited to, hydroxypropyl cellulose ethers (HPC) such as Klucel® orNisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such asE461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® 5100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® 512.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

Liquid formulation dosage forms for oral administration are optionallyaqueous suspensions selected from the group including, but not limitedto, pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).In addition to therapeutic agent the liquid dosage forms optionallyinclude additives, such as: (a) disintegrating agents; (b) dispersingagents; (c) wetting agents; (d) at least one preservative, (e) viscosityenhancing agents, (f) at least one sweetening agent, and (g) at leastone flavoring agent. In some embodiments, the aqueous dispersionsfurther includes a crystal-forming inhibitor.

In some embodiments, the pharmaceutical formulations described hereinare self-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion. SEDDS, as opposed to emulsions or microemulsions,spontaneously form emulsions when added to an excess of water withoutany external mechanical dispersion or agitation. An advantage of SEDDSis that only gentle mixing is required to distribute the dropletsthroughout the solution. Additionally, water or the aqueous phase isoptionally added just prior to administration, which ensures stabilityof an unstable or hydrophobic active ingredient. Thus, the SEDDSprovides an effective delivery system for oral and parenteral deliveryof hydrophobic active ingredients. In some embodiments, SEDDS providesimprovements in the bioavailability of hydrophobic active ingredients.Methods of producing self-emulsifying dosage forms include, but are notlimited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and6,960,563.

Buccal formulations that include a therapeutic agent are administeredusing a variety of formulations known in the art. For example, suchformulations include, but are not limited to, U.S. Pat. Nos. 4,229,447,4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosageforms described herein can further include a bioerodible (hydrolysable)polymeric carrier that also serves to adhere the dosage form to thebuccal mucosa. For buccal or sublingual administration, the compositionsmay take the form of tablets, lozenges, or gels formulated in aconventional manner.

For intravenous injections, a therapeutic agent is optionally formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. For otherparenteral injections, appropriate formulations include aqueous ornonaqueous solutions, preferably with physiologically compatible buffersor excipients.

Parenteral injections optionally involve bolus injection or continuousinfusion. Formulations for injection are optionally presented in unitdosage form, e.g., in ampoules or in multi dose containers, with anadded preservative. In some embodiments, a pharmaceutical compositiondescribed herein is in a form suitable for parenteral injection as asterile suspensions, solutions or emulsions in oily or aqueous vehicles,and contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Pharmaceutical formulations for parenteraladministration include aqueous solutions of an agent that modulates theactivity of a carotid body in water soluble form. Additionally,suspensions of an agent that modulates the activity of a carotid bodyare optionally prepared as appropriate, e.g., oily injectionsuspensions.

Conventional formulation techniques include, e.g., one or a combinationof methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dryor non-aqueous granulation, (5) wet granulation, or (6) fusion. Othermethods include, e.g., spray drying, pan coating, melt granulation,granulation, fluidized bed spray drying or coating (e.g., wurstercoating), tangential coating, top spraying, tableting, extruding and thelike.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

Suitable disintegrants for use in the solid dosage forms describedherein include, but are not limited to, natural starch such as cornstarch or potato starch, a pregelatinized starch, or sodium starchglycolate, a cellulose such as methylcrystalline cellulose,methylcellulose, microcrystalline cellulose, croscarmellose, or across-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose, cross-linked carboxymethylcellulose, orcross-linked croscarmellose, a cross-linked starch such as sodium starchglycolate, a cross-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a gum such as agar, guar, locust bean,Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite,sodium lauryl sulfate, sodium lauryl sulfate in combination starch, andthe like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, hydroxyethylcellulose,hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose,microcrystalline dextrose, amylose, magnesium aluminum silicate,polysaccharide acids, bentonites, gelatin, polyvinylpyrrolidone/vinylacetate copolymer, crospovidone, povidone, starch, pregelatinizedstarch, tragacanth, dextrin, a sugar, such as sucrose, glucose,dextrose, molasses, mannitol, sorbitol, xylitol, lactose, a natural orsynthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapolhusks, starch, polyvinylpyrrolidone, larch arabogalactan, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Binder levels of up to 70% in tablet formulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described hereininclude, for example, e.g., butylated hydroxytoluene (BHT), sodiumascorbate, and tocopherol.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms of the pharmaceutical compositions described herein. The amountsof such additives can be readily determined by one skilled in the art,according to the particular properties desired.

In various embodiments, the particles of a therapeutic agents and one ormore excipients are dry blended and compressed into a mass, such as atablet, having a hardness sufficient to provide a pharmaceuticalcomposition that substantially disintegrates within less than about 30minutes, less than about 35 minutes, less than about 40 minutes, lessthan about 45 minutes, less than about 50 minutes, less than about 55minutes, or less than about 60 minutes, after oral administration,thereby releasing the formulation into the gastrointestinal fluid.

In other embodiments, a powder including a therapeutic agent isformulated to include one or more pharmaceutical excipients and flavors.Such a powder is prepared, for example, by mixing the therapeutic agentand optional pharmaceutical excipients to form a bulk blend composition.Additional embodiments also include a suspending agent and/or a wettingagent. This bulk blend is uniformly subdivided into unit dosagepackaging or multi-dosage packaging units.

In still other embodiments, effervescent powders are also prepared.Effervescent salts have been used to disperse medicines in water fororal administration.

In some embodiments, the pharmaceutical dosage forms are formulated toprovide a controlled release of a therapeutic agent. Controlled releaserefers to the release of the therapeutic agent from a dosage form inwhich it is incorporated according to a desired profile over an extendedperiod of time. Controlled release profiles include, for example,sustained release, prolonged release, pulsatile release, and delayedrelease profiles. In contrast to immediate release compositions,controlled release compositions allow delivery of an agent to a subjectover an extended period of time according to a predetermined profile.Such release rates can provide therapeutically effective levels of agentfor an extended period of time and thereby provide a longer period ofpharmacologic response while minimizing side effects as compared toconventional rapid release dosage forms. Such longer periods of responseprovide for many inherent benefits that are not achieved with thecorresponding short acting, immediate release preparations.

In some embodiments, the solid dosage forms described herein areformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine or large intestine. In one aspect, the enteric coated dosageform is a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. In one aspect, the enteric coated oraldosage form is in the form of a capsule containing pellets, beads orgranules, which include a therapeutic agent that are coated or uncoated.

Any coatings should be applied to a sufficient thickness such that theentire coating does not dissolve in the gastrointestinal fluids at pHbelow about 5, but does dissolve at pH about 5 and above. Coatings aretypically selected from any of the following: Shellac—this coatingdissolves in media of pH>7; Acrylic polymers—examples of suitableacrylic polymers include methacrylic acid copolymers and ammoniummethacrylate copolymers. The Eudragit series E, L, S, RL, RS and NE(Rohm Pharma) are available as solubilized in organic solvent, aqueousdispersion, or dry powders. The Eudragit series RL, NE, and RS areinsoluble in the gastrointestinal tract but are permeable and are usedprimarily for colonic targeting. The Eudragit series E dissolve in thestomach. The Eudragit series L, L-30D and S are insoluble in stomach anddissolve in the intestine; Poly Vinyl Acetate Phthalate (PVAP)-PVAPdissolves in pH>5, and it is much less permeable to water vapor andgastric fluids. Conventional coating techniques such as spray or pancoating are employed to apply coatings. The coating thickness must besufficient to ensure that the oral dosage form remains intact until thedesired site of topical delivery in the intestinal tract is reached.

In other embodiments, the formulations described herein are deliveredusing a pulsatile dosage form. A pulsatile dosage form is capable ofproviding one or more immediate release pulses at predetermined timepoints after a controlled lag time or at specific sites. Exemplarypulsatile dosage forms and methods of their manufacture are disclosed inU.S. Pat. Nos. 5,011,692, 5,017,381, 5,229,135, 5,840,329 and 5,837,284.In one embodiment, the pulsatile dosage form includes at least twogroups of particles, (i.e., multiparticulate) each containing theformulation described herein. The first group of particles provides asubstantially immediate dose of a therapeutic agent upon ingestion by amammal. The first group of particles can be either uncoated or include acoating and/or sealant. In one aspect, the second group of particlescomprises coated particles. The coating on the second group of particlesprovides a delay of from about 2 hours to about 7 hours followingingestion before release of the second dose. Suitable coatings forpharmaceutical compositions are described herein or known in the art.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of a therapeutic agent and at least one dispersingagent or suspending agent for oral administration to a subject. Theformulations may be a powder and/or granules for suspension, and uponadmixture with water, a substantially uniform suspension is obtained.

In some embodiments, particles formulated for controlled release areincorporated in a gel or a patch or a wound dressing.

In one aspect, liquid formulation dosage forms for oral administrationand/or for topical administration as a wash are in the form of aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).In addition to the particles of a therapeutic agent, the liquid dosageforms include additives, such as: (a) disintegrating agents; (b)dispersing agents; (c) wetting agents; (d) at least one preservative,(e) viscosity enhancing agents, (f) at least one sweetening agent, and(g) at least one flavoring agent. In some embodiments, the aqueousdispersions can further include a crystalline inhibitor.

In some embodiments, the liquid formulations also include inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterolesters, taurocholic acid, phosphotidylcholine, oils, such as cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil, and sesameoil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fattyacid esters of sorbitan, or mixtures of these substances, and the like.

Furthermore, pharmaceutical compositions optionally include one or morepH adjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

Additionally, pharmaceutical compositions optionally include one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Other pharmaceutical compositions optionally include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

In one embodiment, the aqueous suspensions and dispersions describedherein remain in a homogenous state, as defined in The USP Pharmacists'Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. In oneembodiment, an aqueous suspension is re-suspended into a homogenoussuspension by physical agitation lasting less than 1 minute. In stillanother embodiment, no agitation is necessary to maintain a homogeneousaqueous dispersion.

Examples of disintegrating agents for use in the aqueous suspensions anddispersions include, but are not limited to, a starch, e.g., a naturalstarch such as corn starch or potato starch, a pregelatinized starch, orsodium starch glycolate; a cellulose such as methylcrystallinecellulose, methylcellulose, croscarmellose, or a cross-linked cellulose,such as cross-linked sodium carboxymethylcellulose, cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose; a cross-linkedstarch such as sodium starch glycolate; a cross-linked polymer such ascrospovidone; a cross-linked polyvinylpyrrolidone; alginate such asalginic acid or a salt of alginic acid such as sodium alginate; a gumsuch as agar, guar, locust bean, Karaya, pectin, or tragacanth; sodiumstarch glycolate; bentonite; a natural sponge; a surfactant; a resinsuch as a cation-exchange resin; citrus pulp; sodium lauryl sulfate;sodium lauryl sulfate in combination starch; and the like.

In some embodiments, the dispersing agents suitable for the aqueoussuspensions and dispersions described herein include, for example,hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone, and the carbohydrate-based dispersing agents suchas, for example, hydroxypropylcellulose and hydroxypropyl celluloseethers, hydroxypropyl methylcellulose and hydroxypropyl methylcelluloseethers, carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,hydroxypropylmethyl-cellulose acetate stearate, noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer,4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers; and poloxamines. Inother embodiments, the dispersing agent is selected from a group notcomprising one of the following agents: hydrophilic polymers;electrolytes; Tween® 60 or 80; PEG; polyvinylpyrrolidone (PVP);hydroxypropylcellulose and hydroxypropyl cellulose ethers; hydroxypropylmethylcellulose and hydroxypropyl methylcellulose ethers;carboxymethylcellulose sodium; methylcellulose; hydroxyethylcellulose;hydroxypropylmethyl-cellulose phthalate; hydroxypropylmethyl-celluloseacetate stearate; non-crystalline cellulose; magnesium aluminumsilicate; triethanolamine; polyvinyl alcohol (PVA);4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde; poloxamers; or poloxamines.

Wetting agents suitable for the aqueous suspensions and dispersionsdescribed herein include, but are not limited to, cetyl alcohol,glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g.,the commercially available Tweens® such as e.g., Tween 20® and Tween80®, and polyethylene glycols, oleic acid, glyceryl monostearate,sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium oleate, sodium lauryl sulfate, sodium docusate,triacetin, vitamin E TPGS, sodium taurocholate, simethicone,phosphotidylcholine and the like.

Suitable preservatives for the aqueous suspensions or dispersionsdescribed herein include, for example, potassium sorbate, parabens(e.g., methylparaben and propylparaben), benzoic acid and its salts,other esters of parahydroxybenzoic acid such as butylparaben, alcoholssuch as ethyl alcohol or benzyl alcohol, phenolic compounds such asphenol, or quaternary compounds such as benzalkonium chloride.Preservatives, as used herein, are incorporated into the dosage form ata concentration sufficient to inhibit microbial growth.

Suitable viscosity enhancing agents for the aqueous suspensions ordispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, Plasdon® S-630, carbomer,polyvinyl alcohol, alginates, acacia, chitosans and combinationsthereof. The concentration of the viscosity enhancing agent will dependupon the agent selected and the viscosity desired.

Examples of sweetening agents suitable for the aqueous suspensions ordispersions described herein include, for example, acacia syrup,acesulfame K, alitame, aspartame, chocolate, cinnamon, citrus, cocoa,cyclamate, dextrose, fructose, ginger, glycyrrhetinate, glycyrrhiza(licorice) syrup, monoammonium glyrrhizinate (MagnaSweet®), malitol,mannitol, menthol, neohesperidine DC, neotame, Prosweet® Powder,saccharin, sorbitol, stevia, sucralose, sucrose, sodium saccharin,saccharin, aspartame, acesulfame potassium, mannitol, sucralose,tagatose, thaumatin, vanilla, xylitol, or any combination thereof.

In some embodiments, a therapeutic agent is prepared as transdermaldosage form. In some embodiments, the transdermal formulations describedherein include at least three components: (1) a therapeutic agent; (2) apenetration enhancer; and (3) an optional aqueous adjuvant. In someembodiments the transdermal formulations include additional componentssuch as, but not limited to, gelling agents, creams and ointment bases,and the like. In some embodiments, the transdermal formulation ispresented as a patch or a wound dressing. In some embodiments, thetransdermal formulation further include a woven or non-woven backingmaterial to enhance absorption and prevent the removal of thetransdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein can maintain a saturated orsupersaturated state to promote diffusion into the skin.

In one aspect, formulations suitable for transdermal administration of atherapeutic agent described herein employ transdermal delivery devicesand transdermal delivery patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive. In one aspect, such patches are constructed for continuous,pulsatile, or on demand delivery of pharmaceutical agents. Stillfurther, transdermal delivery of the therapeutic agents described hereincan be accomplished by means of iontophoretic patches and the like. Inone aspect, transdermal patches provide controlled delivery of atherapeutic agent. In one aspect, transdermal devices are in the form ofa bandage comprising a backing member, a reservoir containing thetherapeutic agent optionally with carriers, optionally a ratecontrolling barrier to deliver the therapeutic agent to the skin of thehost at a controlled and predetermined rate over a prolonged period oftime, and means to secure the device to the skin.

In further embodiments, topical formulations include gel formulations(e.g., gel patches which adhere to the skin). In some of suchembodiments, a gel composition includes any polymer that forms a gelupon contact with the body (e.g., gel formulations comprising hyaluronicacid, pluronic polymers, poly(lactic-co-glycolic acid (PLGA)-basedpolymers or the like). In some forms of the compositions, theformulation comprises a low-melting wax such as, but not limited to, amixture of fatty acid glycerides, optionally in combination with cocoabutter which is first melted. Optionally, the formulations furthercomprise a moisturizing agent.

In certain embodiments, delivery systems for pharmaceutical therapeuticagents may be employed, such as, for example, liposomes and emulsions.In certain embodiments, compositions provided herein can also include anmucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, a therapeutic agent described herein may beadministered topically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical therapeutic agents can contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

An aerosol formulation for nasal administration is generally an aqueoussolution designed to be administered to the nasal passages in drops orsprays. Nasal solutions can be similar to nasal secretions in that theyare generally isotonic and slightly buffered to maintain a pH of about5.5 to about 6.5, although pH values outside of this range canadditionally be used. Antimicrobial agents or preservatives can also beincluded in the formulation.

An aerosol formulation for inhalations and inhalants can be designed sothat the agent or combination of agents is carried into the respiratorytree of the subject when administered by the nasal or oral respiratoryroute. Inhalation solutions can be administered, for example, by anebulizer. Inhalations or insufflations, comprising finely powdered orliquid drugs, can be delivered to the respiratory system as apharmaceutical aerosol of a solution or suspension of the agent orcombination of agents in a propellant, e.g., to aid in disbursement.Propellants can be liquefied gases, including halocarbons, for example,fluorocarbons such as fluorinated chlorinated hydrocarbons,hydrochlorofluorocarbons, and hydrochlorocarbons, as well ashydrocarbons and hydrocarbon ethers.

Halocarbon propellants can include fluorocarbon propellants in which allhydrogens are replaced with fluorine, chlorofluorocarbon propellants inwhich all hydrogens are replaced with chlorine and at least onefluorine, hydrogen-containing fluorocarbon propellants, andhydrogen-containing chlorofluorocarbon propellants. Hydrocarbonpropellants useful include, for example, propane, isobutane, n-butane,pentane, isopentane and neopentane. A blend of hydrocarbons can also beused as a propellant. Ether propellants include, for example, dimethylether as well as the ethers. An aerosol formulation can also comprisemore than one propellant. For example, the aerosol formulation cancomprise more than one propellant from the same class, such as two ormore fluorocarbons; or more than one, more than two, more than threepropellants from different classes, such as a fluorohydrocarbon and ahydrocarbon. Pharmaceutical compositions of the present disclosure canalso be dispensed with a compressed gas, e.g., an inert gas such ascarbon dioxide, nitrous oxide or nitrogen.

Aerosol formulations can also include other components, for example,ethanol, isopropanol, propylene glycol, as well as surfactants or othercomponents such as oils and detergents. These components can serve tostabilize the formulation and/or lubricate valve components.

The aerosol formulation can be packaged under pressure and can beformulated as an aerosol using solutions, suspensions, emulsions,powders and semisolid preparations. For example, a solution aerosolformulation can comprise a solution of an agent such as a transporter,carrier, or ion channel inhibitor in (substantially) pure propellant oras a mixture of propellant and solvent. The solvent can be used todissolve the agent and/or retard the evaporation of the propellant.Solvents can include, for example, water, ethanol and glycols. Anycombination of suitable solvents can be use, optionally combined withpreservatives, antioxidants, and/or other aerosol components.

An aerosol formulation can be a dispersion or suspension. A suspensionaerosol formulation can comprise a suspension of an agent or combinationof agents, e.g., a transporter, carrier, or ion channel inhibitor, and adispersing agent. Dispersing agents can include, for example, sorbitantrioleate, oleyl alcohol, oleic acid, lecithin and corn oil. Asuspension aerosol formulation can also include lubricants,preservatives, antioxidant, and/or other aerosol components.

An aerosol formulation can similarly be formulated as an emulsion. Anemulsion aerosol formulation can include, for example, an alcohol suchas ethanol, a surfactant, water and a propellant, as well as an agent orcombination of agents, e.g., a transporter, carrier, or ion channel. Thesurfactant used can be nonionic, anionic or cationic. One example of anemulsion aerosol formulation comprises, for example, ethanol,surfactant, water and propellant. Another example of an emulsion aerosolformulation comprises, for example, vegetable oil, glyceryl monostearateand propane.

Kits

Disclosed herein, in some embodiments, are kits for using thecompositions described herein. In some embodiments, the kits disclosedherein may be used to treat a disease or disorder in a subject; orselect a subject for treatment and/or monitor a treatment disclosedherein. In some embodiments, the kit comprises the compositionsdescribed herein, which can be used to perform the methods describedherein. Kits comprise an assemblage of materials or components,including at least one of the compositions. Thus, in some embodimentsthe kit contains a composition including of the pharmaceuticalcomposition, for the treatment of an autoimmune disease.

In some instances, the kits described herein comprise components forselecting for the homogenous population of extracellular vesicles. Insome embodiments, the kit comprises the components for assaying thenumber of units of the immune checkpoint moiety expressed on the surfaceof the extracellular vesicle. In some embodiments, the kit comprisescomponents for performing assays such as enzyme-linked immunosorbentassay (ELISA), single-molecular array (Simoa), PCR, and qPCR. The exactnature of the components configured in the kit depends on its intendedpurpose. For example, some embodiments are configured for the purpose oftreating a disease or condition disclosed herein (e.g., autoimmunedisease) in a subject. In some embodiments, the kit is configuredparticularly for the purpose of treating mammalian subjects. In someembodiments, the kit is configured particularly for the purpose oftreating human subjects.

Instructions for use may be included in the kit. Optionally, the kitalso contains other useful components, such as, diluents, buffers,pharmaceutically acceptable carriers, syringes, catheters, applicators,pipetting or measuring tools, bandaging materials or other usefulparaphernalia. The materials or components assembled in the kit can beprovided to the practitioner stored in any convenient and suitable waysthat preserve their operability and utility. For example the componentscan be in dissolved, dehydrated, or lyophilized form; they can beprovided at room, refrigerated or frozen temperatures. The componentsare typically contained in suitable packaging material(s). As employedherein, the phrase “packaging material” refers to one or more physicalstructures used to house the contents of the kit, such as compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in gene expression assays and in the administration oftreatments. As used herein, the term “package” refers to a suitablesolid matrix or material such as glass, plastic, paper, foil, and thelike, capable of holding the individual kit components. Thus, forexample, a package can be a glass vial or prefilled syringes used tocontain suitable quantities of the pharmaceutical composition. Thepackaging material has an external label which indicates the contentsand/or purpose of the kit and its components.

II. Platforms

Described herein, in some embodiments, are platforms for generating theextracellular vesicles described herein. In some embodiments, theplatforms conform to good manufacturing practices (GMP) standard. Insome embodiments, the composition comprising the extracellular vesicleis generated according to good manufacturing practices (GMP). In someembodiments, the composition comprises a pathogen level that issubstantially free of pathogens. In some embodiments, the compositionhas a contaminant level that is substantially free of contaminants. Insome embodiments, the composition comprises low immunogenicity.

In some embodiments, the composition described herein is generated andisolated via hypotonic treatment and centrifugation. In someembodiments, the extracellular vesicles are isolated from mesenchymalstem cells (MSC) expressing the extracellular vesicles primarily byusing hypotonic treatment such that the MSC ruptures and extracellularvesicles are released. In some instances, the MSC are resuspended inhypotonic solution to induce cell swelling. In some embodiments, theplatform comprises phase-contrast microscopy to monitor cell swelling.In some embodiments, the platform comprises a homogenizer to rupture theswollen cells to release extracellular vesicles. In some embodiments,the platform comprises means for separating the ruptured cells in agradient (e.g., a sucrose gradient) to separate out the extracellularvesicles. In some embodiments, the platform comprises other componentsto generate extracellular vesicles other approaches of lysing the MSCsuch as mild sonication, freeze-thaw, French-press, or needle-passaging.In some embodiments, the platform comprises centrifuges to centrifugeand isolate the fraction comprising the extracellular vesicles. In someembodiments, the platform comprises means for separating a fractioncomprising the extracellular vesicle by floatation in a discontinuoussucrose density gradient.

In some embodiments, the platform comprises means for generating theextracellular vesicles by extrusion. In some embodiments, the extrusionprocess separates and isolates the extracellular vesicles based on thesizes or diameters of the extracellular vesicles. Exemplary extrusionprocess comprises the use of membranes with various pore sizes. Themembranes can separate the extracellular vesicles based on the sizes ordiameters of the extracellular vesicles from a solution comprising theruptured MSC. Extracellular vesicles can be further isolated and reducedin size by continued extrusion following extrusion with increasinglysmaller membrane pore sizes, ranging from 150 nm to 10 nm. When thefinal extrusion is complete, extracellular vesicle can be are pelletedby centrifugation. In some embodiments, the platform comprisescomponents for performing sonication, extrusion, highpressure/homogenization, microfluidization, or detergent dialysis.

In some embodiments, the platform comprises components for determiningunit numbers of immune checkpoint moiety per extracellular vesicle.

III. Methods Generating Extracellular Vesicles

Described herein, in some embodiments, are methods of generating thecompositions descried herein utilizing the platforms described herein.In some embodiments, extracellular vesicles can be isolated from orsecreted by a cell. For example, extracellular vesicles can be generatedfrom lysing the cells to release the extracellular vesicles. In somecases, the cells secretes the extracellular vesicles, where theextracellular vesicles can then be isolated.

In some embodiments, the cell for generating the extracellular vesiclecan be from a cell line, stem cells, primary cells, or differentiatedcells. In some embodiments, the extracellular vesicle donor cells can beselected from the group consisting of human embryonic fibroblasts (HEF),dendritic cells, mesenchymal stem cells, bone marrow-derived dendriticcells, bone marrow derived stromal cells, adipose stromal cells,endothelial cells, enucleated cells, neural stem cells, immaturedendritic cells, and immune cells, bone marrow stromal cells, marrowderived adult progenitor cells (MAPCs), endothelial progenitor cells(EPC), blast cells, intermediate progenitor cells formed in thesubventricular zone, neural stem cells, muscle stem cells, satellitecells, liver stem cells, hematopoietic stem cells, bone marrow stromalcells, epidermal stem cells, embryonic stem cells, umbilical cord stemcells, precursor cells, muscle precursor cells, myoblast,cardiomyoblast, neural precursor cells, glial precursor cells, neuronalprecursor cells, or hepatoblasts.

In some embodiments, the cell for generating the extracellular vesiclescan be a genetically modified cell, where a genetic modification moietyis introduced into the modified cell. In some embodiments, at least oneheterologous polynucleotide encoding a transgene is introduced into themodified cell. In some embodiments, the heterologous polynucleotideencodes any one of the immune checkpoint moiety described herein. Insome embodiments, the heterologous polynucleotide encodes any one of thetargeting moiety described herein. In some embodiments, the heterologouspolynucleotide encodes any one of the transmembrane moiety describedherein. In some embodiments, the heterologous polynucleotide encodes anyone of the fusion peptide described herein. In some embodiments, theheterologous polynucleotide encodes any one of the immune evasion moietydescribed herein. In some embodiments, the heterologous polynucleotidecan be integrated into the chromosome of the modified cell. In someembodiments, the heterologous polynucleotide is not integrated into thechromosome of the modified cell.

In some embodiments, the a genetic modification moiety regulates theexpressions of the heterologous polynucleotide. In some embodiments, thea genetic modification moiety increases the expressions of theheterologous polynucleotide. In some embodiments, the geneticmodification moiety comprises a CRISPR-Cas polypeptide. In someembodiments, the genetic modification moiety can be, for example, Class1 CRISPR-associated (Cas) polypeptides, Class 2 Cas polypeptides, type ICas polypeptides, type II Cas polypeptides, type III Cas polypeptides,type IV Cas polypeptides, type V Cas polypeptides, and type VI,CRISPR-associated RNA binding proteins, or a functional fragmentthereof. Cas polypeptides suitable for use with the present disclosurecan include Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (orCas13a), Cas13b, Cas13c, Cas13d, C2C3, Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2,Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG,CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, CsxlO, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cu1966; anyderivative thereof; any variant thereof; or any fragment thereof. Insome embodiments, Cas13 can include, but are not limited to, Cas13a,Cas13b, Cas13c, and Cas 13d (e.g., CasRx). CRISPR/Cas can be DNA and/orRNA cleaving, or can exhibit reduced cleavage activity. Geneticmodification moiety can be configured to complex with at least oneheterologous RNA polynucleotide. In some cases, the genetic modificationmoiety can be fused with a transcription activator or transcriptionrepressor.

Any suitable nuclease (e.g., endonuclease) can be used in as the geneticmodification moiety. Suitable nucleases include, but are not limited to,CRISPR-associated (Cas) proteins or Cas nucleases including type ICRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas)polypeptides, type III CRISPR-associated (Cas) polypeptides, type IVCRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas)polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zincfinger nucleases (ZFN); transcription activator-like effector nucleases(TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associatedRNA binding proteins; recombinases; flippases; transposases; Argonaute(Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute(aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregoryiArgonaute (NgAgo)); Adenosine deaminases acting on RNA (ADAR); CIRT,PUF, homing endonuclease, or any functional fragment thereof, anyderivative thereof; any variant thereof; and any fragment thereof.

A genetic modification moiety as disclosed herein can be coupled (e.g.,linked or fused) to additional peptide sequences which are not involvedin regulating gene expression, for example linker sequences, targetingsequences, etc. The term “targeting sequence,” as used herein, refers toa nucleotide sequence and the corresponding amino acid sequence whichencodes a targeting polypeptide which mediates the localization (orretention) of a protein to a sub-cellular location, e.g., plasmamembrane or membrane of a given organelle, nucleus, cytosol,mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast,peroxisome or other organelle. For example, a targeting sequence candirect a protein (e.g., a receptor polypeptide or an adaptorpolypeptide) to a nucleus utilizing a nuclear localization signal (NLS);outside of a nucleus of a cell, for example to the cytoplasm, utilizinga nuclear export signal (NES); mitochondria utilizing a mitochondrialtargeting signal; the endoplasmic reticulum (ER) utilizing anER-retention signal; a peroxisome utilizing a peroxisomal targetingsignal; plasma membrane utilizing a membrane localization signal; orcombinations thereof.

A genetic modification moiety as disclosed herein can be a part of afusion construct (e.g., a fusion protein). As used herein, “fusion” canrefer to a protein and/or nucleic acid comprising one or more non-nativesequences (e.g., moieties). A fusion can comprise one or more of thesame non-native sequences. A fusion can comprise one or more ofdifferent non-native sequences. A fusion can be a chimera. A fusion cancomprise a nucleic acid affinity tag. A fusion can comprise a barcode. Afusion can comprise a peptide affinity tag. A fusion can provide forsubcellular localization of the site-directed polypeptide (e.g., anuclear localization signal (NLS) for targeting to the nucleus, amitochondrial localization signal for targeting to the mitochondria, achloroplast localization signal for targeting to a chloroplast, anendoplasmic reticulum (ER) retention signal, and the like). A fusion canprovide a non-native sequence (e.g., affinity tag) that can be used totrack or purify. A fusion can be a small molecule such as biotin or adye such as Alexa fluor dyes, Cyanine3 dye, Cyanine5 dye.

A fusion can refer to any protein with a functional effect. For example,a fusion protein can comprise methyltransferase activity, demethylaseactivity, dismutase activity, alkylation activity, depurinationactivity, oxidation activity, pyrimidine dimer forming activity,integrase activity, transposase activity, recombinase activity,polymerase activity (e.g., a reverse transcriptase activity), ligaseactivity, helicase activity, photolyase activity or glycosylaseactivity, acetyltransferase activity, deacetylase activity, kinaseactivity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity, remodelling activity,protease activity, oxidoreductase activity, transferase activity,hydrolase activity, lyase activity, isomerase activity, synthaseactivity, synthetase activity, or demyristoylation activity. An effectorprotein can modify a genomic locus. A fusion protein can be a fusion ina Cas protein. A fusion protein can be a non-native sequence in a Casprotein.

In some embodiments, the genetic modification moiety can be fused to oneor more transcription repressor domains, activator domains, epigeneticdomains, recombinase domains, transposase domains, flippase domains,nickase domains, or any combination thereof. The activator domain caninclude one or more tandem activation domains located at the carboxylterminus of the protein. In some cases, the genetic modification moietyincludes one or more tandem repressor domains located at the carboxylterminus of the protein. Non-limiting exemplary activation domainsinclude GAL4, herpes simplex activation domain VP16, VP64 (a tetramer ofthe herpes simplex activation domain VP16), NF-κB p65 subunit,Epstein-Barr virus R transactivator (Rta) and are described in Chavez etal., Nat Methods, 2015, 12(4):326-328. Non-limiting exemplary repressiondomains include the KRAB (Kruppel-associated box) domain of Kox1, theMad mSIN3 interaction domain (SID), ERF repressor domain (ERD), and aredescribed in Chavez et al., Nat Methods, 2015, 12(4):326-328. In someembodiments, the genetic modification moiety includes one or more tandemrepressor domains located at the amino terminus of the protein.

In some embodiments, the nuclease disclosed herein can be a protein thatlacks nucleic acid cleavage activity. In some cases, a Cas protein is adead Cas protein. A dead Cas protein can be a protein that lacks nucleicacid cleavage activity. A Cas protein can comprise a modified form of awild type Cas protein. The modified form of the wild type Cas proteincan comprise an amino acid change (e.g., deletion, insertion, orsubstitution) that reduces the nucleic acid-cleaving activity of the Casprotein. For example, the modified form of the Cas protein can have lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than5%, or less than 1% of the nucleic acid-cleaving activity of thewild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified formof Cas protein can have no substantial nucleic acid-cleaving activity.When a Cas protein is a modified form that has no substantial nucleicacid-cleaving activity, it can be referred to as enzymatically inactiveand/or “dead” (abbreviated by “d”). A dead Cas protein (e.g., dCas,dCas9) can bind to a target polynucleotide but may not cleave the targetpolynucleotide. In some aspects, a dead Cas protein is a dead Cas9protein.

In some embodiments, a dCas (e.g., dCas9) polypeptide can associate witha single guide RNA (sgRNA) to activate or repress transcription oftarget DNA. sgRNAs can be introduced into cells expressing theengineered chimeric receptor polypeptide. In some cases, such cellscontain one or more different sgRNAs that target the same nucleic acid.In other cases, the sgRNAs target different nucleic acids in the cell.

In some embodiments, the genetic modification moiety can comprise acatalytically inactive Cas polypeptide, where the nuclease activity ofthe Cas polypeptide is eliminated or substantially eliminated.

In some instances, the genetic modification moiety can comprise acatalytically inactivated Cas9 (dCas9), any derivative thereof; anyvariant thereof; or any fragment thereof.

In some instances, the genetic modification moiety can comprise acatalytically inactivated Cas12 (dCas12), any derivative thereof; anyvariant thereof; or any fragment thereof.

In some instances, the genetic modification moiety can comprise acatalytically inactivated Cas13 (dCas13); any derivative thereof; anyvariant thereof or any fragment thereof.

In some embodiments, the genetic modification moiety can be complexedwith the at least one heterologous polynucleotide as described herein.In some embodiments, the at least one heterologous polynucleotide can beeither heterologous DNA polynucleotide or heterologous RNApolynucleotide. In some embodiments, the genetic modification moiety canbe complexed with at least one heterologous RNA polynucleotide. In someembodiments, the complexing with the at least one heterologous RNApolynucleotide direct and target the genetic modification moiety to theportion of the heterologous polynucleotide.

In some cases, the compositions and methods described herein comprise atleast one heterologous polynucleotide. In some cases, the compositionsand methods described herein comprise a plurality of heterologousnucleic acids. In some embodiments, the polynucleotide can bedeoxyribonucleic acid (DNA). In some cases, the DNA sequence can besingle-stranded or doubled-stranded. In some embodiments, the at leastone heterologous nucleic acid polynucleotide can be ribonucleic acid(RNA).

In some embodiments, the genetic modification moiety can be complexedwith the at least one heterologous RNA polynucleotide. The at least oneheterologous RNA polynucleotide can comprise a nucleic-acid targetingregion that comprises a complementary sequence to a nucleic acidsequence of the heterologous polynucleotide that encodes any one of themoieties described herein for specificity of the genetic modificationmoiety-dependent targeting. In some embodiments, the at least oneheterologous RNA polynucleotide can be guide nucleic acid (or guide RNA)comprising two separate nucleic acid molecules, which can be referred toas a double guide nucleic acid or a single nucleic acid molecule, whichcan be referred to as a single guide nucleic acid (e.g., sgRNA). In someembodiments, the guide nucleic acid is a single guide nucleic acidcomprising a fused CRISPR RNA (crRNA) and a transactivating crRNA(tracrRNA). In some embodiments, the guide nucleic acid is a singleguide nucleic acid comprising a crRNA. In some embodiments, the guidenucleic acid is a single guide nucleic acid comprising a crRNA butlacking a tracRNA. In some embodiments, the guide nucleic acid is adouble guide nucleic acid comprising non-fused crRNA and tracrRNA. Anexemplary double guide nucleic acid can comprise a crRNA-like moleculeand a tracrRNA-like molecule. An exemplary single guide nucleic acid cancomprise a crRNA-like molecule. An exemplary single guide nucleic acidcan comprise a fused crRNA-like molecule and a tracrRNA-like molecule.

A crRNA can comprise the nucleic acid-targeting segment (e.g., spacerregion) of the guide nucleic acid and a stretch of nucleotides that canform one half of a double-stranded duplex of the Cas protein-bindingsegment of the guide nucleic acid.

A tracrRNA can comprise a stretch of nucleotides that forms the otherhalf of the double-stranded duplex of the Cas protein-binding segment ofthe gRNA. A stretch of nucleotides of a crRNA can be complementary toand hybridize with a stretch of nucleotides of a tracrRNA to form thedouble-stranded duplex of the Cas protein-binding domain of the guidenucleic acid.

The crRNA and tracrRNA can hybridize to form a guide nucleic acid. ThecrRNA can also provide a single-stranded nucleic acid targeting segment(e.g., a spacer region) that hybridizes to a target nucleic acidrecognition sequence (e.g., protospacer). The sequence of a crRNA,including spacer region, or tracrRNA molecule can be designed to bespecific to the species in which the guide nucleic acid is to be used.

In some embodiments, the nucleic acid-targeting region of a guidenucleic acid can be between 18 to 72 nucleotides in length. The nucleicacid-targeting region of a guide nucleic acid (e.g., spacer region) canhave a length of from about 12 nucleotides to about 100 nucleotides. Forexample, the nucleic acid-targeting region of a guide nucleic acid(e.g., spacer region) can have a length of from about 12 nucleotides(nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 ntto about 40 nt, from about 12 nt to about 30 nt, from about 12 nt toabout 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about19 nt, from about 12 nt to about 18 nt, from about 12 nt to about 17 nt,from about 12 nt to about 16 nt, or from about 12 nt to about 15 nt.Alternatively, the DNA-targeting segment can have a length of from about18 nt to about 20 nt, from about 18 nt to about 25 nt, from about 18 ntto about 30 nt, from about 18 nt to about 35 nt, from about 18 nt toabout 40 nt, from about 18 nt to about 45 nt, from about 18 nt to about50 nt, from about 18 nt to about 60 nt, from about 18 nt to about 70 nt,from about 18 nt to about 80 nt, from about 18 nt to about 90 nt, fromabout 18 nt to about 100 nt, from about 20 nt to about 25 nt, from about20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 ntto about 40 nt, from about 20 nt to about 45 nt, from about 20 nt toabout 50 nt, from about 20 nt to about 60 nt, from about 20 nt to about70 nt, from about 20 nt to about 80 nt, from about 20 nt to about 90 nt,or from about 20 nt to about 100 nt. The length of the nucleicacid-targeting region can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30 or more nucleotides. The length of the nucleicacid-targeting region (e.g., spacer sequence) can be at most 5, 10, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

In some embodiments, the nucleic acid-targeting region of a guidenucleic acid (e.g., spacer) is 20 nucleotides in length. In someembodiments, the nucleic acid-targeting region of a guide nucleic acidis 19 nucleotides in length. In some embodiments, the nucleicacid-targeting region of a guide nucleic acid is 18 nucleotides inlength. In some embodiments, the nucleic acid-targeting region of aguide nucleic acid is 17 nucleotides in length. In some embodiments, thenucleic acid-targeting region of a guide nucleic acid is 16 nucleotidesin length. In some embodiments, the nucleic acid-targeting region of aguide nucleic acid is 21 nucleotides in length. In some embodiments, thenucleic acid-targeting region of a guide nucleic acid is 22 nucleotidesin length.

The nucleotide sequence of the guide nucleic acid that is complementaryto a nucleotide sequence (target sequence) of the target nucleic acidcan have a length of, for example, at least about 12 nt, at least about15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt,at least about 25 nt, at least about 30 nt, at least about 35 nt or atleast about 40 nt. The nucleotide sequence of the guide nucleic acidthat is complementary to a nucleotide sequence (target sequence) of thetarget nucleic acid can have a length of from about 12 nucleotides (nt)to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt toabout 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt,from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, fromabout 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 ntto about 40 nt, from about 19 nt to about 45 nt, from about 19 nt toabout 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt,from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, fromabout 20 nt to about 50 nt, or from about 20 nt to about 60 nt.

A protospacer sequence of a targeted polynucleotide can be identified byidentifying a PAM within a region of interest and selecting a region ofa desired size upstream or downstream of the PAM as the protospacer. Acorresponding spacer sequence can be designed by determining thecomplementary sequence of the protospacer region.

A spacer sequence can be identified using a computer program (e.g.,machine readable code). The computer program can use variables such aspredicted melting temperature, secondary structure formation, andpredicted annealing temperature, sequence identity, genomic context,chromatin accessibility, % GC, frequency of genomic occurrence,methylation status, presence of SNPs, and the like.

The percent complementarity between the nucleic acid-targeting sequence(e.g., a spacer sequence of the at least one heterologous polypeptide asdisclosed herein) and the target nucleic acid (e.g., a protospacersequence of the heterologous polynucleotide encoding any one of themoieties described herein) can be at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%, or 100%. The percentcomplementarity between the nucleic acid-targeting sequence and thetarget nucleic acid can be at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or 100% over about 20 contiguous nucleotides.

The Cas protein-binding segment of a guide nucleic acid can comprise twostretches of nucleotides (e.g., crRNA and tracrRNA) that arecomplementary to one another. The two stretches of nucleotides (e.g.,crRNA and tracrRNA) that are complementary to one another can becovalently linked by intervening nucleotides (e.g., a linker in the caseof a single guide nucleic acid). The two stretches of nucleotides (e.g.,crRNA and tracrRNA) that are complementary to one another can hybridizeto form a double stranded RNA duplex or hairpin of the Casprotein-binding segment, thus resulting in a stem-loop structure. ThecrRNA and the tracrRNA can be covalently linked via the 3′ end of thecrRNA and the 5′ end of the tracrRNA. Alternatively, tracrRNA and crRNAcan be covalently linked via the 5′ end of the tracrRNA and the 3′ endof the crRNA.

The Cas protein binding segment of a guide nucleic acid can have alength of from about 10 nucleotides to about 100 nucleotides, e.g., fromabout 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt,from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, fromabout 70 nt to about 80 nt, from about 80 nt to about 90 nt, or fromabout 90 nt to about 100 nt. For example, the Cas protein-bindingsegment of a guide nucleic acid can have a length of from about 15nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, fromabout 15 nt to about 40 nt, from about 15 nt to about 30 nt or fromabout 15 nt to about 25 nt.

The dsRNA duplex of the Cas protein-binding segment of the guide nucleicacid can have a length from about 6 base pairs (bp) to about 50 bp. Forexample, the dsRNA duplex of the protein-binding segment can have alength from about 6 bp to about 40 bp, from about 6 bp to about 30 bp,from about 6 bp to about 25 bp, from about 6 bp to about 20 bp, fromabout 6 bp to about 15 bp, from about 8 bp to about 40 bp, from about 8bp to about 30 bp, from about 8 bp to about 25 bp, from about 8 bp toabout 20 bp or from about 8 bp to about 15 bp. For example, the dsRNAduplex of the Cas protein-binding segment can have a length from aboutfrom about 8 bp to about 10 bp, from about 10 bp to about 15 bp, fromabout 15 bp to about 18 bp, from about 18 bp to about 20 bp, from about20 bp to about 25 bp, from about 25 bp to about 30 bp, from about 30 bpto about 35 bp, from about 35 bp to about 40 bp, or from about 40 bp toabout 50 bp.

In some embodiments, the dsRNA duplex of the Cas protein-binding segmentcan have a length of 36 base pairs. The percent complementarity betweenthe nucleotide sequences that hybridize to form the dsRNA duplex of theprotein-binding segment can be at least about 60%. For example, thepercent complementarity between the nucleotide sequences that hybridizeto form the dsRNA duplex of the protein-binding segment can be at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%. In some cases, the percentcomplementarity between the nucleotide sequences that hybridize to formthe dsRNA duplex of the protein-binding segment is 100%.

The linker (e.g., that links a crRNA and a tracrRNA in a single guidenucleic acid) can have a length of from about 3 nucleotides to about 100nucleotides. For example, the linker can have a length of from about 3nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt) to about80 nt, from about 3 nucleotides (nt) to about 70 nt, from about 3nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) to about50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about20 nt or from about 3 nucleotides (nt) to about 10 nt. For example, thelinker can have a length of from about 3 nt to about 5 nt, from about 5nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt toabout 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt,from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, fromabout 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about80 nt to about 90 nt, or from about 90 nt to about 100 nt. In someembodiments, the linker of a DNA-targeting RNA is 4 nt.

Guide nucleic acids of the disclosure can include modifications orsequences that provide for additional desirable features (e.g., modifiedor regulated stability; subcellular targeting; tracking with afluorescent label; a binding site for a protein or protein complex; andthe like). Examples of such modifications include, for example, a 5′ cap(a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail (a 3′ poly(A)tail); a riboswitch sequence (e.g., to allow for regulated stabilityand/or regulated accessibility by proteins and/or protein complexes); astability control sequence; a sequence that forms a dsRNA duplex (ahairpin)); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, and so forth); a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyl transferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and combinations thereof.

A guide nucleic acid can comprise one or more modifications (e.g., abase modification, a backbone modification), to provide the nucleic acidwith a new or enhanced feature (e.g., improved stability). A guidenucleic acid can comprise a nucleic acid affinity tag. A nucleoside canbe a base-sugar combination. The base portion of the nucleotide can be aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides can benucleosides that further include a phosphate group covalently linked tothe sugar portion of the nucleoside. For those nucleosides that includea pentofuranosyl sugar, the phosphate group can be linked to the 2′, the3′, or the 5′ hydroxyl moiety of the sugar. In forming guide nucleicacids, the phosphate groups can covalently link adjacent nucleosides toone another to form a linear polymeric compound. In turn, the respectiveends of this linear polymeric compound can be further joined to form acircular compound; however, linear compounds can be suitable. Inaddition, linear compounds can have internal nucleotide basecomplementarity and can therefore fold in a manner as to produce a fullyor partially double-stranded compound. Further, within guide nucleicacids, the phosphate groups can commonly be referred to as forming theinternucleoside backbone of the guide nucleic acid. The linkage orbackbone of the guide nucleic acid can be a 3′ to 5′ phosphodiesterlinkage.

A guide nucleic acid can comprise a modified backbone and/or modifiedinternucleoside linkages. Modified backbones can include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone.

Suitable modified guide nucleic acid backbones containing a phosphorusatom therein can include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as3′-alkylene phosphonates, 5′-alkylene phosphonates, chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates,and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs,and those having inverted polarity wherein one or more internucleotidelinkages is a 3′ to 3′, a 5′ to 5′ or a 2′ to 2′ linkage. Suitable guidenucleic acids having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage (such as a singleinverted nucleoside residue in which the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (e.g., potassiumchloride or sodium chloride), mixed salts, and free acid forms can alsobe included.

A guide nucleic acid can comprise one or more phosphorothioate and/orheteroatom internucleoside linkages, in particular —CH2-NH—O—CH2-,—CH2-N(CH3)-O—CH2- (a methylene (methylimino) or MMI backbone),—CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and —O—N(CH3)-CH2-CH2-(wherein the native phosphodiester internucleotide linkage isrepresented as —O—P(═O)(OH)—O—CH2-).

A guide nucleic acid can comprise a morpholino backbone structure. Forexample, a nucleic acid can comprise a 6-membered morpholino ring inplace of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

A guide nucleic acid can comprise polynucleotide backbones that areformed by short chain alkyl or cycloalkyl internucleoside linkages,mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, orone or more short chain heteroatomic or heterocyclic internucleosidelinkages. These can include those having morpholino linkages (formed inpart from the sugar portion of a nucleoside); siloxane backbones;sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH2 component parts.

A guide nucleic acid can comprise a nucleic acid mimetic. The term“mimetic” can be intended to include polynucleotides wherein only thefuranose ring or both the furanose ring and the internucleotide linkageare replaced with non-furanose groups, replacement of only the furanosering can also be referred as being a sugar surrogate. The heterocyclicbase moiety or a modified heterocyclic base moiety can be maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backboneof a polynucleotide can be replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleotides can beretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. The backbone in PNA compounds cancomprise two or more linked aminoethylglycine units which gives PNA anamide containing backbone. The heterocyclic base moieties can be bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

A guide nucleic acid can comprise linked morpholino units (morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring.Linking groups can link the morpholino monomeric units in a morpholinonucleic acid. Non-ionic morpholino-based oligomeric compounds can haveless undesired interactions with cellular proteins. Morpholino-basedpolynucleotides can be non-ionic mimics of guide nucleic acids. Avariety of compounds within the morpholino class can be joined usingdifferent linking groups. A further class of polynucleotide mimetic canbe referred to as cyclohexenyl nucleic acids (CeNA). The furanose ringnormally present in a nucleic acid molecule can be replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can beprepared and used for oligomeric compound synthesis usingphosphoramidite chemistry. The incorporation of CeNA monomers into anucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements withsimilar stability to the native complexes. A further modification caninclude Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group islinked to the 4′ carbon atom of the sugar ring thereby forming a2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.The linkage can be a methylene (—CH2-), group bridging the 2′ oxygenatom and the 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs candisplay very high duplex thermal stabilities with complementary nucleicacid (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradationand good solubility properties.

A guide nucleic acid can comprise one or more substituted sugarmoieties. Suitable polynucleotides can comprise a sugar substituentgroup selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl;O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10alkenyl and alkynyl. Particularly suitable are O((CH2)nO) mCH3,O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, andO(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. A sugarsubstituent group can be selected from: C1 to C10 lower alkyl,substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkarylor O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2,NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an guide nucleic acid, or a group for improving thepharmacodynamic properties of an guide nucleic acid, and othersubstituents having similar properties. A suitable modification caninclude 2′-methoxyethoxy (2′-O—CH2 CH2OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE, an alkoxyalkoxy group). A furthersuitable modification can include 2′-dimethylaminooxyethoxy, (aO(CH2)2ON(CH3)2 group, also known as 2′-DMAOE), and2′-dimethylaminoethoxyethoxy (also known as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), 2′-O—CH2-O—CH2-N(CH3)2.

Other suitable sugar substituent groups can include methoxy (—O—CH3),aminopropoxy CH2 CH2NH2), allyl (—CH2-CH═CH2), —O-allyl CH2-CH═CH2) andfluoro (F). 2′-sugar substituent groups can be in the arabino (up)position or ribo (down) position. A suitable 2′-arabino modification is2′-F. Similar modifications can also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked nucleotides and the 5′ positionof 5′ terminal nucleotide. Oligomeric compounds can also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

A guide nucleic acid can also include nucleobase (or “base”)modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases can include the purine bases, (e.g., adenine (A)and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine(C) and uracil (U)). Modified nucleobases can include other syntheticand natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH3) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino¬adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modifiednucleobases can include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H¬pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties can include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases can be useful for increasing the binding affinity of apolynucleotide compound. These can include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions can increase nucleic acid duplexstability by 0.6-1.2° C. and can be suitable base substitutions (e.g.,when combined with 2′-O-methoxyethyl sugar modifications).

A modification of a guide nucleic acid can comprise chemically linkingto the guide nucleic acid one or more moieties or conjugates that canenhance the activity, cellular distribution or cellular uptake of theguide nucleic acid. These moieties or conjugates can include conjugategroups covalently bound to functional groups such as primary orsecondary hydroxyl groups. Conjugate groups can include, but are notlimited to, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that can enhance thepharmacokinetic properties of oligomers. Conjugate groups can include,but are not limited to, cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties include groups that improve uptake, enhanceresistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that can enhance thepharmacokinetic properties include groups that improve uptake,distribution, metabolism or excretion of a nucleic acid. Conjugatemoieties can include but are not limited to lipid moieties such as acholesterol moiety, cholic acid a thioether, (e.g.,hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g.,dodecandiol or undecyl residues), a phospholipid (e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

Described herein, in some embodiments, are heterologous RNApolynucleotides comprising crRNAs. In some embodiments, the cRNAcomprises targeting sequence that is complementary to the heterologouspolynucleotide encoding any one of the moieties described herein.

In some embodiments, the crRNA comprises 5 nt to 100 nt. In someembodiments, the crRNA comprises 5 nt to 6 nt, 5 nt to 7 nt, 5 nt to 8nt, 5 nt to 9 nt, 5 nt to 10 nt, 5 nt to 15 nt, 5 nt to 20 nt, 5 nt to25 nt, 5 nt to 50 nt, 5 nt to 100 nt, 6 nt to 7 nt, 6 nt to 8 nt, 6 ntto 9 nt, 6 nt to 10 nt, 6 nt to 15 nt, 6 nt to 20 nt, 6 nt to 25 nt, 6nt to 50 nt, 6 nt to 100 nt, 7 nt to 8 nt, 7 nt to 9 nt, 7 nt to 10 nt,7 nt to 15 nt, 7 nt to 20 nt, 7 nt to 25 nt, 7 nt to 50 nt, 7 nt to 100nt, 8 nt to 9 nt, 8 nt to 10 nt, 8 nt to 15 nt, 8 nt to 20 nt, 8 nt to25 nt, 8 nt to 50 nt, 8 nt to 100 nt, 9 nt to 10 nt, 9 nt to 15 nt, 9 ntto 20 nt, 9 nt to 25 nt, 9 nt to 50 nt, 9 nt to 100 nt, 10 nt to 15 nt,10 nt to 20 nt, 10 nt to 25 nt, 10 nt to 50 nt, 10 nt to 100 nt, 15 ntto 20 nt, 15 nt to 25 nt, 15 nt to 50 nt, 15 nt to 100 nt, 20 nt to 25nt, 20 nt to 50 nt, 20 nt to 100 nt, 25 nt to 50 nt, 25 nt to 100 nt, or50 nt to 100 nt. In some embodiments, the crRNA comprises 5 nt, 6 nt, 7nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In someembodiments, the crRNA comprises at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt,10 nt, 15 nt, 20 nt, 25 nt, or 50 nt. In some embodiments, the crRNAcomprises at most 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50nt, or 100 nt. In some embodiments, the crRNA comprises at least 5 nt to100 nt. In some embodiments, the crRNA comprises at least 5 nt to 6 nt,5 nt to 7 nt, 5 nt to 8 nt, 5 nt to 9 nt, 5 nt to 10 nt, 5 nt to 15 nt,5 nt to 20 nt, 5 nt to 25 nt, 5 nt to 50 nt, 5 nt to 100 nt, 6 nt to 7nt, 6 nt to 8 nt, 6 nt to 9 nt, 6 nt to 10 nt, 6 nt to 15 nt, 6 nt to 20nt, 6 nt to 25 nt, 6 nt to 50 nt, 6 nt to 100 nt, 7 nt to 8 nt, 7 nt to9 nt, 7 nt to 10 nt, 7 nt to 15 nt, 7 nt to 20 nt, 7 nt to 25 nt, 7 ntto 50 nt, 7 nt to 100 nt, 8 nt to 9 nt, 8 nt to 10 nt, 8 nt to 15 nt, 8nt to 20 nt, 8 nt to 25 nt, 8 nt to 50 nt, 8 nt to 100 nt, 9 nt to 10nt, 9 nt to 15 nt, 9 nt to 20 nt, 9 nt to 25 nt, 9 nt to 50 nt, 9 nt to100 nt, 10 nt to 15 nt, 10 nt to 20 nt, 10 nt to 25 nt, 10 nt to 50 nt,10 nt to 100 nt, 15 nt to 20 nt, 15 nt to 25 nt, 15 nt to 50 nt, 15 ntto 100 nt, 20 nt to 25 nt, 20 nt to 50 nt, 20 nt to 100 nt, 25 nt to 50nt, 25 nt to 100 nt, or 50 nt to 100 nt. In some embodiments, the crRNAcomprises at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25nt, 50 nt, or 100 nt. In some embodiments, the crRNA comprises at leastat least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, or 50nt. In some embodiments, the crRNA comprises at least at most 6 nt, 7nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt. In someembodiments, the crRNA comprises at most 5 nt to 100 nt. In someembodiments, the crRNA comprises at most 5 nt to 6 nt, 5 nt to 7 nt, 5nt to 8 nt, 5 nt to 9 nt, 5 nt to 10 nt, 5 nt to 15 nt, 5 nt to 20 nt, 5nt to 25 nt, 5 nt to 50 nt, 5 nt to 100 nt, 6 nt to 7 nt, 6 nt to 8 nt,6 nt to 9 nt, 6 nt to 10 nt, 6 nt to 15 nt, 6 nt to 20 nt, 6 nt to 25nt, 6 nt to 50 nt, 6 nt to 100 nt, 7 nt to 8 nt, 7 nt to 9 nt, 7 nt to10 nt, 7 nt to 15 nt, 7 nt to 20 nt, 7 nt to 25 nt, 7 nt to 50 nt, 7 ntto 100 nt, 8 nt to 9 nt, 8 nt to 10 nt, 8 nt to 15 nt, 8 nt to 20 nt, 8nt to 25 nt, 8 nt to 50 nt, 8 nt to 100 nt, 9 nt to 10 nt, 9 nt to 15nt, 9 nt to 20 nt, 9 nt to 25 nt, 9 nt to 50 nt, 9 nt to 100 nt, 10 ntto 15 nt, 10 nt to 20 nt, 10 nt to 25 nt, 10 nt to 50 nt, 10 nt to 100nt, 15 nt to 20 nt, 15 nt to 25 nt, 15 nt to 50 nt, 15 nt to 100 nt, 20nt to 25 nt, 20 nt to 50 nt, 20 nt to 100 nt, 25 nt to 50 nt, 25 nt to100 nt, or 50 nt to 100 nt. In some embodiments, the crRNA comprises atmost 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or100 nt. In some embodiments, the crRNA comprises at most at least 5 nt,6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 25 nt, or 50 nt. In someembodiments, the crRNA comprises at most at most 6 nt, 7 nt, 8 nt, 9 nt,10 nt, 15 nt, 20 nt, 25 nt, 50 nt, or 100 nt.

In some embodiments, the genetic modification moiety and theheterologous polynucleotide can be delivered into the cell via the useof expression vectors. In the context of an expression vector, thevector can be readily introduced into a host cell, e.g., mammalian,bacterial, yeast, or insect cell by any method in the art. For example,the expression vector can be transferred into a host cell by physical,chemical, or biological means. In some embodiments, the geneticmodification moiety and the heterologous polynucleotide can be deliveredinto the cell via physical methods such as calcium phosphateprecipitation, lipofection, particle bombardment, microinjection, genegun, electroporation, and the like. Methods for producing cellscomprising vectors and/or exogenous nucleic acids are suitable formethods herein (see, e.g., Sambrook et al., 2012, Molecular Cloning: ALaboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). Onemethod for the introduction of a polynucleotide into a host cell iscalcium phosphate transfection. In some embodiments, the geneticmodification moiety and the heterologous polynucleotide can be deliveredinto the cell via biological methods such as the use of DNA and RNAvectors. Viral vectors, and especially retroviral vectors, have becomethe most widely used method for inserting genes into mammalian, e.g.,human cells. Other viral vectors, in some embodiments, are derived fromlentivirus, poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like. Exemplary viral vectors includeretroviral vectors, adenoviral vectors, adeno-associated viral vectors(AAVs), pox vectors, parvoviral vectors, baculovirus vectors, measlesviral vectors, or herpes simplex virus vectors (HSVs). In someinstances, the retroviral vectors include gamma-retroviral vectors suchas vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV,MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In someinstances, the retroviral vectors also include lentiviral vectors suchas those derived from the human immunodeficiency virus (HIV) genome. Insome instances, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7,AAV8, or AAV9 serotype. In some instances, viral vector is a chimericviral vector, comprising viral portions from two or more viruses. Inadditional instances, the viral vector is a recombinant viral vector. Insome embodiments, the genetic modification moiety and the heterologouspolynucleotide can be delivered into the cell via chemical means such asmacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. An exemplary colloidal system for use as adelivery vehicle in vitro and in vivo is a liposome (e.g., an artificialmembrane vesicle). Other methods of state-of-the-art targeted deliveryof nucleic acids are available, such as delivery of polynucleotides withtargeted nanoparticles or other suitable sub-micron sized deliverysystem. In some embodiments, the genetic modification moiety and theheterologous polynucleotide can be delivered into the cell via anon-viral delivery system. Non-viral delivery system can be liposome.The use of lipid formulations is contemplated for the introduction ofthe nucleic acids into a host cell (in vitro, ex vivo or in vivo). Inanother aspect, the nucleic acid is associated with a lipid. The nucleicacid associated with a lipid, in some embodiments, is encapsulated inthe aqueous interior of a liposome, interspersed within the lipidbilayer of a liposome, attached to a liposome via a linking moleculethat is associated with both the liposome and the oligonucleotide,entrapped in a liposome, complexed with a liposome, dispersed in asolution containing a lipid, mixed with a lipid, combined with a lipid,contained as a suspension in a lipid, contained or complexed with amicelle, or otherwise associated with a lipid. Lipid, lipid/DNA orlipid/expression vector associated compositions are not limited to anyparticular structure in solution. For example, in some embodiments, theyare present in a bilayer structure, as micelles, or with a “collapsed”structure. Alternately, they are simply be interspersed in a solution,possibly forming aggregates that are not uniform in size or shape.Lipids are fatty substances which are, in some embodiments, naturallyoccurring or synthetic lipids. For example, lipids include the fattydroplets that naturally occur in the cytoplasm as well as the class ofcompounds which contain long-chain aliphatic hydrocarbons and theirderivatives, such as fatty acids, alcohols, amines, amino alcohols, andaldehydes. Lipids suitable for use are obtained from commercial sources.For example, in some embodiments, dimyristyl phosphatidylcholine(“DMPC”) is obtained from Sigma, St. Louis, Mo.; in some embodiments,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Choi”), in some embodiments, isobtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol(“DMPG”) and other lipids are often obtained from Avanti Polar Lipids,Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform orchloroform/methanol are often stored at about −20° C. Chloroform is usedas the only solvent since it is more readily evaporated than methanol.“Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes are often characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh etal., 1991 Glycobiology 5: 505-10). However, compositions that havedifferent structures in solution than the normal vesicular structure arealso encompassed. For example, the lipids, in some embodiments, assume amicellar structure or merely exist as nonuniform aggregates of lipidmolecules. Also contemplated are lipofectamine-nucleic acid complexes.In some embodiments, the genetic modification moiety and theheterologous polynucleotide can be delivered into the cell can bepackaged and delivered to the cell via extracellular vesicles. Theextracellular vesicles can be any membrane-bound particles. In someembodiments, the extracellular vesicles can be any membrane-boundparticles secreted by at least one cell. In some instances, theextracellular vesicles can be any membrane-bound particles synthesizedin vitro. In some instances, the extracellular vesicles can be anymembrane-bound particles synthesized without a cell. In some cases, theextracellular vesicles can be exosomes, microvesicles, retrovirus-likeparticles, apoptotic bodies, apoptosomes, oncosomes, exophers, envelopedviruses, exomeres, or other very large extracellular vesicles.

Identifying and Isolating Homogenous Population of ExtracellularVesicles

Described herein, in some embodiments, are methods for utilizing theplatforms described herein to generate compositions comprising ahomogeneous population of extracellular vesicles. In some embodiments,the method identifies and isolates the homogenous population ofextracellular vesicles based on the dimensions (e.g. diameters or sizes)of the extracellular vesicles. In some embodiments, the methodidentifies and isolates the homogenous population of extracellularvesicles based on the mass of the extracellular vesicles. In someembodiments, the method identifies and isolates the homogeneouspopulation of extracellular vesicles based on the number of units ofimmune checkpoint moiety encapsulated, secreted, or expressed on thesurface of the extracellular vesicle. In some embodiments, the methodidentifies and isolates the homogenous population of extracellularvesicles based on a combination of the dimensions and the number ofunits if immune checkpoint moiety encapsulated, secreted, or expressedon the surface of the extrasellar vesicle. In some embodiments, themethod identifies and isolates the homogenous population ofextracellular vesicles based on a combination of dimensions and thenumber of unit of immune checkpoint moiety expressed on the surface ofthe extracellular vesicle.

In some embodiments, the method of identifying and isolating thehomogenous population of extracellular vesicles comprises performingdifferential ultracentrifugation to isolate a homogenous population ofextracellular vesicle based on density. In some embodiments the methodcomprises performing filtration or ultrafiltration to isolate homogenouspopulation of extracellular vesicles based on weights or sizes. In someembodiments, the method comprises performing HPLC. In some embodiments,the method comprises performing extracellular vesicle precipitation,where water-excluding polymers such as polyethylene glycol (PEG) can tieup water molecules and force less soluble components out of solution. Assuch, the precipitate containing extracellular vesicle cam ne isolatedby means of either low-speed centrifugation or filtration. In someembodiments, the method comprises performing affinity-based capture bycapturing the extracellular vesicles by immunoaffinity. Examples ofproteins or epitope displayed on the surface of the extracellularvesicles include CD9, CD63. CD81. Alix, caveolin-1, CD41, CD4,flotillin, Rab5, HSC70, and Lamp-3. In some embodiments, the methodcomprises performing microfluidics-based isolation method forextracellular vesicle for identifying and isolating a homogenouspopulation of extracellular vesicle based on size, density, andimmunoaffinity, innovative sorting mechanisms such as acoustic,electrophoretic and electromagnetic manipulations can be implemented.With the use of such devices, significant reductions in sample volume,reagent consumption, and isolation time are expected.

In some embodiments, the method of identifying and isolating thehomogenous population of extracellular vesicles comprises basing on thenumber of immune checkpoint moiety expressed on the surface of theextracellular vesicle. In some embodiments, the method comprisesimmunoassay, where antibody recognizing the immune checkpoint moiety isused. In some embodiments, the antibody is conjugated to a detectablemoiety. In some embodiments, the signal detected from the antibodyrecognizing and binding to the immune checkpoint moiety correlates withthe number of immune checkpoint moiety expressed on the surface of theextracellular vesicle. Exemplary detectable moiety includes an enzymaticmoiety (e.g., horseradish peroxidase (HRP), beta-galactosidase, alkalinephosphatase, etc), fluorescent dye, luminescent moiety, radioactivemoiety, colorimetric label, colored latex particle or nanoparticle, andmetal-conjugated moiety such as metallic nanolayer, metallicnanoparticle, or metallic nanoshell-conjugated moiety. In someembodiments, the detectable moiety is directly or indirectly tagged fora colorimetric assay (e.g., for detection of HRP or beta-galactosidaseactivity), visual inspection using light microscopy, immunofluorescencemicroscopy, confocal microscopy, by flow cytometry (FACS),autoradiography electron microscopy, immunostaining, or subcellularfractionation.

In some embodiments, the method of identifying and isolating thehomogenous population of extracellular vehicles comprises identifyingand isolating the homogenous population of extracellular vesicles basedon both diameter and number of units of immune checkpoint moietyexpressed on the surface of the extracellular vesicle. For example, themethod identifies and isolates a homogenous population of extracellularvesicles comprising a diameter of about 50 nm and about 2000 units ofimmune checkpoint moiety expressed on the surface of the extracellularvesicles. In some embodiments, the method identifies and isolates ahomogenous population of extracellular vesicles comprising a diameter ofabout 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70, nm, 80 nm, 90 nm,100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm,190 nm, 200 nm, or more and 500 units, 1000 units, 1500 units, 2000units, 2500 units, 3000 units, 3500 units, 4000 units, 4500 units, 5000units, 5500 units, 6000 units, 7500 units, 8000 units, 8500 units, 9000units, 9500 units, 10000 units, 11000 nuts, 12000 units, 13000 units,14000 units, 15000 units, or more of the immune checkpoint expressed onthe surface of the extracellular vesicle.

Treatment

Disclosed herein, in some embodiments, are methods of treating a diseaseor a disorder in a subject, comprising administrating of therapeuticeffective amount of the compositions or pharmaceutical compositionsdescribed herein to the subject. In some embodiments, the disease ordisorder is an autoimmune disease, including Rheumatoid arthritis,Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus,Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn'sdisease, Graves' disease, Juvenile arthritis, Lyme disease chronic,Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerativecolitis (UC), Uveitis, Inflammatory eye conditions, Vitiligo, COPD,complication from Organ transplantation, or graft-versus-host disease.

In some embodiments, the method comprises steps of: contacting the cellwith the compositions or pharmaceutical compositions as describedherein; upon said contacting, the immune checkpoint moiety is deliveredto the target cell. In some embodiments, the immune checkpoint moietymodulates immune response or of the target cell. In some embodiments,the contacting occurs in vivo, ex vivo, or in vitro. In some embodiment,the composition or pharmaceutical composition can directly beadministered to the subject.

In some embodiments, the composition or pharmaceutical composition canbe administered to the subject alone (e.g., standalone treatment). Insome embodiments, the composition is administered in combination with anadditional agent. In some embodiments, the composition is a first-linetreatment for the disease or condition. In some embodiments, thecomposition is a second-line, third-line, or fourth-line treatment, forthe autoimmune disease.

In general, methods disclosed herein comprise administering acomposition by oral administration. However, in some instances, methodscomprise administering a composition by intraperitoneal injection. Insome instances, methods comprise administering a composition in the formof an anal suppository. In some instances, methods compriseadministering a composition by intravenous (“i.v.”) administration. Itis conceivable that one can also administer compositions disclosedherein by other routes, such as subcutaneous injection, intramuscularinjection, intradermal injection, transdermal injection percutaneousadministration, intranasal administration, intralymphatic injection,rectal administration intragastric administration, or any other suitableparenteral administration. In some embodiments, routes for localdelivery closer to site of injury or inflammation are preferred oversystemic routes. Routes, dosage, time points, and duration ofadministrating therapeutics can be adjusted. In some embodiments,administration of therapeutics is prior to, or after, onset of either,or both, acute and chronic symptoms of the disease or condition.

An effective dose and dosage of the compositions to prevent or treat theautoimmune diseases herein is defined by an observed beneficial responserelated to the autoimmune disease or condition, or symptom of theautoimmune disease. In some instances, the beneficial response comprisesreduction of symptoms of autoimmune disease. Additional beneficialresponse comprises preventing, alleviating, arresting, or curing theautoimmune disease. In instances where the composition is nottherapeutically effective or is not providing a sufficient alleviationof the disease or condition, or symptom of the disease or condition,then the dosage amount and/or route of administration can be changed, oran additional agent can be administered to the subject, along with thecomposition. In some embodiments, as a patient is started on a regimenof a composition, the patient is also weaned off (e.g., step-wisedecrease in dose) a second treatment regimen.

Suitable dose and dosage administrated to a subject is determined byfactors including, but no limited to, the particular composition,disease condition and its severity, the identity (e.g., weight, sex,age) of the subject in need of treatment, and can be determinedaccording to the particular circumstances surrounding the case,including, e.g., the specific agent being administered, the route ofadministration, the condition being treated, and the subject beingtreated.

In some embodiments, the administration of the composition is hourly,once every 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours 22hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4years, or 5 years, or 10 years. The effective dosage ranges can beadjusted based on subject's response to the treatment. Some routes ofadministration will require higher concentrations of effective amount oftherapeutics than other routes.

In certain embodiments, where the patient's condition does not improve,upon the doctor's discretion the administration of composition isadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease or condition. In certain embodiments wherein a patient's statusdoes improve, the dose of composition being administered can betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In specific embodiments, the length ofthe drug holiday is between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, or more than 28 days. The dosereduction during a drug holiday is, by way of example only, by 10%-100%,including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In certainembodiments, the dose of drug being administered can be temporarilyreduced or temporarily suspended for a certain length of time (i.e., a“drug diversion”). In specific embodiments, the length of the drugdiversion is between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, or more than 28 days. The dose reductionduring a drug diversion is, by way of example only, by 10%-100%,including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After asuitable length of time, the normal dosing schedule is optionallyreinstated.

In some embodiments, once improvement of the patient's conditions hasoccurred, a maintenance dose is administered if necessary. Subsequently,in specific embodiments, the dosage or the frequency of administration,or both, is reduced, as a function of the symptoms, to a level at whichthe improved disease, disorder or condition is retained. In certainembodiments, however, the patient requires intermittent treatment on along-term basis upon any recurrence of symptoms.

Toxicity and therapeutic efficacy of such therapeutic regimens aredetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 and the ED50. The dose ratio between the toxic andtherapeutic effects is the therapeutic index and it is expressed as theratio between LD50 and ED50. In certain embodiments, the data obtainedfrom cell culture assays and animal studies are used in formulating thetherapeutically effective daily dosage range and/or the therapeuticallyeffective unit dosage amount for use in mammals, including humans. Insome embodiments, the daily dosage amount of the composition describedherein lies within a range of circulating concentrations that includethe ED50 with minimal toxicity. In certain embodiments, the daily dosagerange and/or the unit dosage amount varies within this range dependingupon the dosage form employed and the route of administration utilized.

A composition can be used alone or in combination with an additionalagent. In some cases, an “additional agent” as used herein isadministered alone. The composition and the additional agent can beadministered together or sequentially. The combination therapies can beadministered within the same day, or can be administered one or moredays, weeks, months, or years apart. Examples of additional agent caninclude other immune modulators such as antibodies targeting cytokinesor small molecules.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

EXAMPLES

The following illustrative examples are representative of embodiments ofthe stimulation, systems, and methods described herein and are not meantto be limiting in any way.

Example 1. Mesenchymal Stem Cells (MSCs) and their CorrespondentExtracellular Vesicles (EVs) Comprise PD-L1

The presence of PD-L1 on MSCs and their correspondent extracellularvesicles was confirmed by western blotting. As shown in FIG. 1, a clearband with size of 45 kDa was detected in MSCs and correspondentexosomes. Starvation media was used as negative control and isletexosomes was used as positive control. To standardize these results, thesame assay was performed on the MSC EVs from three different donors ofumbilical cord MSCs.

MSC lystate and MSC EV samples were prepared by sonication with volumesof MSC lystate and MSC EV samples corresponding to 25 μg protein. Thesesamples were analyzed using a gradient precast polyacrylamide gel(Mini-PROTEAN; Bio-Rad laboratories, Hercules, Calif., USA). The sampleswere then transferred onto a nitrocellulose membrane which was blockedusing 5% blotting grade Blocker Non-Fat Dry Milk (Bio-Rad Laboratories)in Tris-buffer saline (TBS) for 2 h. The membrane was subsequentlyincubated with primary antibodies against Purified anti-human CD274(B7-H1, PD-L1) Antibody (Biolegend; cat #329701) in 0.25% blotting gradeBlocker Non-Fat Dry Milk in TBS-Tween (TBST) overnight at 4° C. Themembrane was then washed with TBST for 10 min, three times. Secondaryantibodies ECL anti mouse IgG horseradish peroxidase-linked F(ab′)2fragment diluted in 0.25% blotting grade Blocker Non-Fat Dry Milk inTBST were incubated with the membrane for 1.5 h. The membranes werefinally analyzed with ECL Prime Western Blotting Detection (GEHealthcare) and a VersaDoc 4000 MP (Bio-Rad Laboratories).

Example 2. MSC EVs Suppress CD25+CD8+ Immune Cells

Significant CD25+CD8+ immune cell suppression was observed in humanperipheral blood mononuclear cells (PBMCs) stimulated with IL-2 (500U/ml) for 6 days. It was also observed that this suppression waslessened in experimental groups wherein the PBMCs were also contactedwith PD-L1 inhibiting antibodies. A schematic for this experiment isrepresented in FIG. 2.

The representative flow cytometry plots of FIG. 3 show that in thepresence of blocking PD-L1, the suppressive activity of MSC EVs towardsCD8+CD25+ population significantly decreases. Further, while the controlgroup comprising isotype control antibodies shows the same suppressiveactivity similar to MSC EVs alone. These results show that suppressiveactivity of MSC EVs is primarily a function of the presence of PD-L1.

Example 3. MSCs Genetically Modified to Produce More Exosomal PD-L1 thanWildtype MSCs

MSCs are harvested from the umbilical cord of healthy donors. Onceharvested, these MSCs are cultured for further experimentation.Transmembrane CD63 is targeted for genetic modification by knowntechniques (e.g. CRISPR/Cas9). The extracellular domain of PD-L1 isfused to CD63. Several specific subsets of the amino acid sequence ofCD63 have been identified as particularly advantageous regions of PD-L1fusion: directly following RDKVMSE; directly following NNNFRQQ; anddirectly following YPKNNHT. These portions of CD63 are found in theLarge Extracellular Loop (LEL) of CD63. 1, 2, 3, or more G4S linkers areused to fuse the extracellular domain of PD-L1 to CD63.

Following genetic modification of the MSCs, the genetically modifiedMSCS are cultured. Samples of genetically modified MSC lystate andgenetically modified MSC EVs are prepared following the protocol ofExample 1. These samples are analyzed by western blot analysis to testfor the presence of PD-L1. Clear bands with size of 45 kDa are detectedin genetically modified MSCs and their correspondent exosomes. Thesebands are darker in color than the western blot analysis of Example 1,indicating that the genetically modified MSCs and their correspondentexosomes produce more PD-L1 than wildtype MSCs and EVs produced fromwildtype MSCs. Starvation media was used as negative control and isletexosomes was used as positive control.

Example 4. MSCs Genetically Modified to Produce Exosomal ImmuneCheckpoint Inhibitors

The method of Example 3 is utilized to transcribe any immune checkpointinhibitor selected from VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2(CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14),Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2(Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1),Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL,CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160,Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRPalpha (CD172a), or a combination thereof.

Western blot analysis is conducted on the genetically modified MSCs andtheir correspondent EVs to test for the presence of the selected immunecheckpoint inhibitor.

Example 5. Generation/Purification of Potent Genetically Modified MSCsand their Correspondent EVs

The genetically modified MSCs and their correspondent EVs from Example 3are assayed to generate potent MSCs and their correspondent EVssufficient for increased CD25+CD8+ immune cell suppression as comparedto the results of Example 2.

The genetically modified MSCs and their correspondent EVs from Example 3are prepared in a solution comprising anti-PD-L1 antibodies conjugatedwith detectable labels and soluble PD-1. The soluble PD-1 binds toexosomal PD-L1. Due to the interactions between the soluble PD-1 and theexosomal PD-L1 present on the surface of the MSCs and theircorrespondent EVs, the anti-PD-L1 antibodies conjugated with detectablelabels selectively bind to the MSCs and EVs in the sample that expressthe higher levels of exosomal PD-L1 as compared to the MSCs and EVswherein the correspondent exosomal PD-L1 is occupied by the soluble PD-1present in the sample.

Using this assay, potent MSCs and EVs are generated/purified from thesample.

Example 6. The Potent Genetically Modified MSCs and their CorrespondentEVs Show Enhanced CD25+CD8+ Immune Cell Suppression

The potent MSCs and EVs from Example 5 are assayed in the experiment ofExample 2. The experimental groups comprising the potent MSCs and EVsshow significantly increased CD25+CD8+ immune cell suppression comparedto the results of FIG. 3.

Example 7. Potent Genetically Modified MSCs and their Correspondent EVsfor Treatment of Rheumatoid Arthritis

Pharmaceutical compositions comprising the potent EVs from Examples 5and 6 and pharmaceutically acceptable excipients are prepared to treatpatients suffering from rheumatoid arthritis. The pharmaceuticalcompositions comprise between about 10{circumflex over ( )}⁶ to about10{circumflex over ( )}⁸ EVs or between about 1 μg to about 700 mg ofEVs.

The pharmaceutical compositions are administered to patients sufferingfrom rheumatoid arthritis at the site of inflammation. The increasedCD25+CD8+ immune cell suppression of the EVs is sufficient for treatingrheumatoid arthritis.

Example 8. Potent Genetically Modified MSCs and their Correspondent EVsfor Treatment of Graft-Versus-Host Disease in a Patient Undergoing aKidney Transplant

Pharmaceutical compositions comprising the potent EVs from Examples 5and 6 and pharmaceutically acceptable excipients are prepared to treatpatients suffering from graft-versus-host disease in a patientundergoing a kidney transplant. The pharmaceutical compositions comprisebetween about 10{circumflex over ( )}⁶ to about 10{circumflex over ( )}⁸EVs or between about 1 μg to about 700 mg of EVs.

The pharmaceutical compositions are administered to patients sufferingfrom graft-versus-host disease in a patient undergoing a kidneytransplant at the site of inflammation. The increased CD25+CD8+ immunecell suppression of the EVs is sufficient for treating graft-versus-hostdisease in a patient undergoing a kidney transplant.

While the foregoing disclosure has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of thedisclosure. For example, all the techniques and apparatus describedabove can be used in various combinations. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication, patent, patentapplication, and/or other document were individually and separatelyindicated to be incorporated by reference for all purposes.

1. A composition comprising an extracellular vesicle, said extracellularvesicle comprising: a. an immune checkpoint moiety, wherein said immunecheckpoint moiety comprises VISTA, PD-L1, CTLA-4, or any combinationthereof; and b. a transmembrane moiety, wherein said transmembranemoiety comprises CD63, wherein said CD63 comprises 3 transmembranedomains; wherein said immune checkpoint moiety is coupled to saidtransmembrane moiety.
 2. The composition of any one of the precedingclaims, wherein said immune checkpoint moiety is coupled to anextracellular loop of said CD63 to generate a modified CD63.
 3. Thecomposition of any one of the preceding claims, wherein saidextracellular loop is a large extracellular loop or the secondextracellular loop of said modified CD63.
 4. The composition of any oneof the preceding claims, wherein said modified CD63 comprises an aminoacid sequence at least 90% identical to any one of the amino acidsequences set forth in SEQ ID NOS: 6-17.
 5. The composition of any oneof the preceding claims, wherein said modified CD63 comprises an aminoacid sequence at least 95% identical to any one of the amino acidsequences set forth in SEQ ID NOS: 6-17.
 6. The composition of any oneof the preceding claims, wherein said modified CD63 comprises any one ofthe amino acid sequences set forth in SEQ ID NOS: 6-17.
 7. A compositioncomprising an extracellular vesicle, said extracellular vesiclecomprising: a. an immune checkpoint moiety, wherein said immunecheckpoint moiety comprises PD-L1; and b. a transmembrane moiety,wherein said transmembrane moiety comprises lactadherin; wherein saidimmune checkpoint moiety is coupled to said transmembrane moiety.
 8. Acomposition comprising an extracellular vesicle, said extracellularvesicle comprising: a. an immune checkpoint moiety, wherein said immunecheckpoint moiety comprises V-domain Ig suppressor of T cell activation(VISTA), PD-L1, CTLA-4, or any combination thereof; and b. atransmembrane moiety, wherein said transmembrane moiety comprisesglycosylphosphatidylinositol (GPI); wherein said immune checkpointmoiety is coupled to said transmembrane moiety.
 9. A compositioncomprising an extracellular vesicle, said extracellular vesiclecomprising at least one of: a. an immune checkpoint moiety; and b. atransmembrane moiety.
 10. The composition of any one of the previousclaims, wherein the immune checkpoint moiety is encapsulated by theextracellular vesicle.
 11. The composition of any one of the previousclaims, wherein the immune checkpoint moiety is expressed on a surfaceof the extracellular vesicle.
 12. The composition of any one of theprevious claims, wherein the immune checkpoint moiety is secreted by theextracellular vesicle.
 13. The composition of any one of the previousclaims, wherein the immune checkpoint moiety is complexed with thetransmembrane moiety.
 14. The composition of any one of the previousclaims, wherein the immune checkpoint moiety is covalently connectedwith the transmembrane moiety.
 15. The composition of any one of theprevious claims, wherein the immune checkpoint moiety comprises VISTA,PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2,B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1(CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1,PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2,CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70,PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R(PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), or acombination thereof.
 16. The composition of any one of the previousclaims, wherein the immune checkpoint moiety comprises VISTA, PD-L1,CTLA-4, or a combination thereof.
 17. The composition of any one of theprevious claims, wherein the immune checkpoint moiety comprises PD-L1.18. The composition of any one of the previous claims, wherein thetransmembrane moiety is selected from a group consisting of 14-3-3protein zeta/delta, 4-3-3 protein epsilon, 78 kDa glucose-regulatedprotein, acetylcholinesterase/AChE-S, AChE-E, actin, cytoplasmic 1(ACTA), ADAM10, alkaline phosphatase, alpha-enolase, alpha-synuclein,aminopeptidase N, amyloid beta A4/APP, annexin 5A, annexin A2, AP-1,ATF3, ATP citrate lyase, ATPase, beta actin (ACTB), beta-amyloid 42,caveolin-1, CD10, CD11a, CD11b, CD11c, CD14, CD142, CD146, CD163, CD24,CD26/DPP4, CD29/ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49,CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudin,claudin-1 cofilin-1, complement-binding proteins CD55 and CD59,cytosolic heat shock protein 90 alpha, cytosolic heat shock protein 90beta, EBV LMP1, EBV LMP2A, EF-1alpha-1, EF2, EFGR EGFR VIII,emmprin/CD147, enolase 1 alpha (ENO1), EPCAM, ERBB2, tetraspanins (CD9,CD63 and CD81), fatty acid synthase, fetuin-A, flotillin-1, flotillin-2,fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5′nucleotidase,GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 andHSP90), heparan sulfate proteoglycans, heparinase, heterotrimeric Gproteins, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax,huntingtin, ICAM1, integrins, lactadherin, LAMP1/2, leucine-richreceptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associatedmembrane glycoprotein 1, lysosome-associated membrane glycoprotein 2,MEW class I, MHC class II, MUC1, multidrug resistance-associatedprotein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP/Alix,PECAM1, phosphoglycerate kinase, placental prion proteins,prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a,Rab-5b, Rab-5c, Rab-7, Rap 1B, resistin, sonic hedgehog (SHH),surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor(TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin-8,tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein,TYRP-2, vacuolar-sorting protein 35, or zeta polypeptide (YWHAZ). 19.The composition of any one of the previous claims, wherein thetransmembrane moiety comprises lactadherin.
 20. The composition of anyone of the previous claims, wherein the transmembrane moiety comprisesLAMP2 or a variation thereof or a fragment thereof, said LAMP2 is atleast 70% identical to a peptide sequence of SEQ ID NO: 4
 21. Thecomposition of any one of the previous claims, wherein the transmembranemoiety comprises CD63 or a variation thereof or a fragment thereof, saidCD63 is at least 70% identical to a peptide sequence of SEQ ID NO:5. 22.The composition of claim 21, wherein the CD63 is a modified CD63. 23.The composition of claim 22, wherein the modified CD63 is a truncatedCD63.
 24. The composition of claim 22, wherein the modified CD63 ismodified to comprise at least one additional CD63 transmembrane domain.25. The composition of claim 22, wherein the modified CD63 comprises 1transmembrane domain.
 26. The composition of claim 22, wherein themodified CD63 comprises 2 transmembrane domains.
 27. The composition ofclaim 22, wherein the modified CD63 comprises 3 transmembrane domains.28. The composition of claim 22, wherein the modified CD63 comprises 4transmembrane domains.
 29. The composition of claim 22, wherein themodified CD63 comprises 5 transmembrane domains.
 30. The composition ofany one of the previous claims, wherein the immune checkpoint moiety iscomplexed with the modified CD63 at an extracellular loop of themodified CD63.
 31. The composition of any one of the previous claims,wherein the immune checkpoint moiety is complexed with the modified CD63at a large extracellular loop of the modified CD63.
 32. The compositionof any one of the previous claims, wherein the composition furthercomprises a targeting moiety.
 33. The composition of claim 32, whereinthe targeting moiety comprises a peptide that targets cytokine.
 34. Thecomposition of claim 32 or 33, wherein the targeting moiety comprises apeptide that targets a cancer cell marker.
 35. The composition of anyone of the previous claims, wherein the composition further comprises afusogenic moiety.
 36. The composition of claim 35, wherein the fusogenicmoiety comprises a viral fusogenic moiety.
 37. The composition of claim35 or 36, wherein the fusogenic moiety comprises a mammalian fusogenicmoiety.
 38. The composition of any one of the previous claims, whereinthe composition further comprises an immune evasion moiety.
 39. Thecomposition of claim 38 wherein the immune evasion moiety comprisesCD47.
 40. The composition of any one of the previous claims does notcomprise enucleated cell.
 41. The composition of any one of the previousclaims, wherein the extracellular vesicle comprises exosome,microvesicle, retrovirus-like particle, apoptotic body, apoptosome,oncosome, exopher, enveloped virus, exomere, or other very largeextracellular vesicle.
 42. The composition of any one of the previousclaims, wherein the extracellular vesicle comprises exosome.
 43. Thecomposition of any one of the previous claims, wherein the extracellularvesicle comprises a plurality of immune checkpoint moieties.
 44. Thecomposition of claim 43, wherein the extracellular vesicle comprises atleast 10,000 units of the immune checkpoint moiety per eachextracellular vesicle, said extracellular vesicle comprises a diameterof 100 nm.
 45. The composition of claim 43, wherein the extracellularvesicle comprises at least 9,000 units of the immune checkpoint moietyper each extracellular vesicle, said extracellular vesicle comprises adiameter of 100 nm.
 46. The composition of claim 43, wherein theextracellular vesicle comprises at least 8,000 units of the immunecheckpoint moiety per each extracellular vesicle, said extracellularvesicle comprises a diameter of 100 nm.
 47. The composition of claim 43,wherein the extracellular vesicle comprises at least 7,000 units of theimmune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 100 nm.
 48. Thecomposition of claim 43, wherein the extracellular vesicle comprises atleast 6,000 units of the immune checkpoint moiety per each extracellularvesicle, said extracellular vesicle comprises a diameter of 100 nm. 49.The composition of claim 43, wherein the extracellular vesicle comprisesat least 5,000 units of the immune checkpoint moiety per eachextracellular vesicle, said extracellular vesicle comprises a diameterof 100 nm.
 50. The composition of claim 43, wherein the extracellularvesicle comprises at least 3,000 units of the immune checkpoint moietyper each extracellular vesicle, said extracellular vesicle comprises adiameter of 50 nm.
 51. The composition of claim 43, wherein theextracellular vesicle comprises at least 2,500 units of the immunecheckpoint moiety per each extracellular vesicle, said extracellularvesicle comprises a diameter of 50 nm.
 52. The composition of claim 43,wherein the extracellular vesicle comprises at least 2,000 units of theimmune checkpoint moiety per each extracellular vesicle, saidextracellular vesicle comprises a diameter of 50 nm.
 53. The compositionof claim 43, wherein the extracellular vesicle comprises at least 1,500units of the immune checkpoint moiety per each extracellular vesicle,said extracellular vesicle comprises a diameter of 50 nm.
 54. Thecomposition of any one of the previous claims, wherein the compositioncomprises a plurality of exosome.
 55. The composition of any one of theprevious claims, wherein the composition comprises a therapeuticallyeffective amount of exosome.
 56. The composition of claim 54 or 55,comprising at least 10{circumflex over ( )}6 exosomes.
 57. Thecomposition of claim 54 or 55, comprising at least 10{circumflex over( )}7 exosomes.
 58. The composition of claim 54 or 55, comprising atleast 10{circumflex over ( )}8 exosomes.
 59. The composition of claim 54or 55, comprising at least 1 μg of exosomes.
 60. The composition ofclaim 54 or 55, comprising at least 10 μg of exosomes.
 61. Thecomposition of claim 54 or 55, comprising at least 20 μg of exosomes.62. The composition of claim 54 or 55, comprising at least 50 μg ofexosomes.
 63. The composition of claim 54 or 55, comprising at least 100μg of exosomes.
 64. The composition of claim 54 or 55, comprising atleast 150 μg of exosomes.
 65. The composition of claim 54 or 55,comprising at least 200 μg of exosomes.
 66. The composition of claim 54or 55, comprising at least 250 μg of exosomes.
 67. The composition ofclaim 54 or 55, comprising at least 500 μg of exosomes.
 68. Thecomposition of claim 54 or 55, comprising at least 750 μg of exosomes.69. The composition of claim 54 or 55, comprising at least 1 mg ofexosomes.
 70. The composition of claim 54 or 55, comprising at least 2mg of exosomes.
 71. The composition of claim 54 or 55, comprising atleast 3 mg of exosomes.
 72. The composition of claim 54 or 55,comprising at least 4 mg of exosomes.
 73. The composition of claim 54 or55, comprising at least 5 mg of exosomes.
 74. The composition of claim54 or 55, comprising at least 6 mg of exosomes.
 75. The composition ofclaim 54 or 55, comprising at least 7 mg of exosomes.
 76. Thecomposition of claim 54 or 55, comprising at least 100 mg of exosomes.77. The composition of claim 54 or 55, comprising at least 200 mg ofexosomes.
 78. The composition of claim 54 or 55, comprising at least 300mg of exosomes.
 79. The composition of claim 54 or 55, comprising atleast 400 mg of exosomes.
 80. The composition of claim 54 or 55,comprising at least 500 mg of exosomes.
 81. The composition of claim 54or 55, comprising at least 600 mg of exosomes.
 82. The composition ofclaim 54 or 55, comprising at least 700 mg of exosomes.
 83. Thecomposition of any one of the previous claims, wherein the compositionis derived from a cell.
 84. The composition of any one of the previousclaims, wherein the composition is cryopreserved.
 85. The composition ofany one of the previous claims, wherein the composition is lyophilized.86. The composition of any one of the previous claims, wherein thecomposition is stable at 37° C. for 24 hours.
 87. The composition of anyone of the previous claims, wherein the composition is stable at 37° C.for 48 hours.
 88. The composition of any one of the previous claims,wherein the composition is stable at 37° C. for 72 hours.
 89. A cellconfigured to generate extracellular vesicle of any one of claims 1-88.90. The cell of claim 89, wherein said cell is a stem cell.
 91. The cellof claim 89, wherein the cell is a human cell.
 92. The cell of claim 89,wherein the cell is a non-human cell.
 93. The cell of any one of claims89 to 92, wherein the cell is a mesenchymal stem cell.
 94. The cell ofany one of claims 89 to 93, wherein the cell is a genetically modifiedcell.
 95. A method of purifying extracellular vesicle configured toexpress one or more immune checkpoint moiety, said method comprising: a.obtaining a heterogenous population of extracellular vesicle; b.subjecting the heterogenous population of extracellular vesicle to adetection assay solution, said detection assay solution comprising adetecting moiety for complexing with the immune checkpoint moiety; andc. detecting a signal generated from complex formed between the immunecheckpoint moiety and the detecting moiety, wherein intensity of thesignal is proportional to units of immune checkpoint moiety expressed;and d. isolating a subpopulation of the extracellular vesicle based onthe intensity of the signal.
 96. The method of claim 95, wherein thedetecting moiety comprises an antibody.
 97. The method of claim 96,wherein the detecting moiety comprises anti-VISTA antibody, anti-PD-L1antibody, anti-CTLA-4 or a combination thereof.
 98. The method of claim95, wherein the detecting moiety comprises a ligand of the immunecheckpoint moiety.
 99. The method of claim 95, wherein the detectionassay solution further comprises a peptide configured to bind to saidimmune checkpoint moiety.
 100. The method of claim 99, wherein thepeptide configured to bind to said immune checkpoint moiety comprisesPD-1, CD80, CD86, or a combination thereof.
 101. A pharmaceuticalcomposition comprising the composition of any one of claims 1-88. 102.The pharmaceutical composition of claim 101 comprising apharmaceutically acceptable carrier.
 103. The pharmaceutical compositionof claim 101 comprising at least one additional active agent.
 104. Thepharmaceutical composition of claim 101, wherein the pharmaceuticalcomposition is formulated for administering intrathecally,intraocularly, intravitreally, retinally, intravenously,intramuscularly, intraventricularly, intracerebrally, intracerebellarly,intracerebroventricularly, intraperenchymally, subcutaneously, or acombination thereof.
 105. A method of treating an autoimmune disease,the method comprising administering the pharmaceutical composition ofany one of claims 101 to
 104. 106. The method of claim 105, wherein saidautoimmune disease is Rheumatoid arthritis, Systemic lupuserythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis,Inflammatory bowel disease, Celiac disease, Crohn's disease, Graves'disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis,Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC),Uveitis, Inflammatory eye conditions, Vitiligo, COPD, complication fromOrgan transplantation, or graft-versus-host disease.
 107. The method ofclaim 106, wherein said autoimmune disease is Rheumatoid arthritis. 108.A method of suppressing CD8+CD25+ cells in a patient in need thereof,the method comprising administering the composition of any one of claims1-88.
 109. A kit comprising the pharmaceutical composition of claims101-104.
 110. A platform comprising components for generating thecomposition of any one of claims 1-88.